Plant colonization assays using natural microbial barcodes

ABSTRACT

The present disclosure is drawn to methods of utilizing nucleic acid barcodes and corresponding amplifying sites in cells in which the barcodes naturally occur. These barcodes and amplifying sites are reconfigured into a single nucleic acid cassette that provides for ease of use in tagging particular species, strains, or variants of cells, each with a different barcode. These barcodes can be used to track the colonization capabilities of the barcoded cells. The present disclosure further provides for assays that utilize natural barcodes to measure relative microbial colonization ability of a plant root system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application Ser. No. 62/906,419, filed Sep. 26, 2019, and U.S. Provisional Application Ser. No. 62/789,332, filed Jan. 7, 2019, each of which is herein incorporated by reference in its entirety for all purposes.

STATEMENT REGARDING SEQUENCE LISTING

The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing filename: PIVO_012_02WO_SeqList_ST25.txt, date created, Jan. 4, 2020, file size≈594 kilobytes.

BACKGROUND OF THE DISCLOSURE

By 2050 the United Nations' Food and Agriculture Organization projects that total food production must increase by 70% to meet the needs of a growing population, a challenge that is exacerbated by numerous factors, including: diminishing freshwater resources, increasing competition for arable land, rising energy prices, increasing input costs, and the likely need for crops to adapt to the pressures of a drier, hotter, and more extreme global climate.

Current agricultural practices are not well equipped to meet this growing demand for food production, while simultaneously balancing the environmental impacts that result from increased agricultural intensity.

One of the major agricultural inputs needed to satisfy global food demand is nitrogen fertilizer. However, the current industrial standard utilized to produce nitrogen fertilizer, is an artificial nitrogen fixation method called the Haber-Bosch process, which converts atmospheric nitrogen (N₂) to ammonia (NH₃) by a reaction with hydrogen (H₂) using a metal catalyst under high temperatures and pressures. This process is resource intensive and deleterious to the environment.

In contrast to the synthetic Haber-Bosch process, certain biological systems have evolved to fix atmospheric nitrogen. These systems utilize an enzyme called nitrogenase that catalyzes the reaction between N₂ and H₂, and results in nitrogen fixation. For example, rhizobia are diazotrophic bacteria that fix nitrogen after becoming established inside root nodules of legumes. An important goal of nitrogen fixation research is the extension of this phenotype to non-leguminous plants, particularly to important agronomic grasses such as wheat, rice, and corn. However, despite the significant progress made in understanding the development of the nitrogen-fixing symbiosis between rhizobia and legumes, the path to use that knowledge to induce nitrogen-fixing nodules on non-leguminous crops is still not clear.

Consequently, the vast majority of modern row crop agriculture utilizes nitrogen fertilizer that is produced via the resource intensive and environmentally deleterious Haber-Bosch process. For instance, the USDA indicates that the average U.S. corn farmer typically applies between 130 and 200 lb. of nitrogen per acre (146 to 224 kg-Tia). This nitrogen is not only produced in a resource intensive synthetic process, but is applied by heavy machinery crossing/impacting the field's soil, burning petroleum, and requiring hours of human labor.

Furthermore, the nitrogen fertilizer produced by the industrial Haber-Bosch process is not well utilized by the target crop. Rain, runoff, heat, volatilization, and the soil microbiome degrade the applied chemical fertilizer. This equates to not only wasted money, but also adds to increased pollution instead of harvested yield. To this end, the United Nations has calculated that nearly 80% of fertilizer is lost before a crop can utilize it. Consequently, modern agricultural fertilizer production and delivery is not only deleterious to the environment, but it is extremely inefficient.

In order to meet the world's growing food supply needs—while also balancing resource utilization and providing minimal impacts upon environmental systems—a better approach to nitrogen fixation and delivery to plants is urgently needed.

SUMMARY OF THE DISCLOSURE

In some aspects, the disclosure is drawn to a method of barcoding a host cell, the method comprising, (a) obtaining a donor cell; (b) selecting and isolating a first nucleotide sequence in the genome of the donor cell, selecting and isolating a second nucleotide sequence in the genome of the donor cell, and selecting and isolating a third nucleotide sequence in the genome of the donor cell; (c) creating a native barcode polynucleotide cassette comprising the first, second, and third nucleotide sequences oriented 5′ to 3′ as: (i) the first nucleotide sequence, wherein the first nucleotide sequence is a forward primer binding site, (ii) the second nucleotide sequence, wherein the second nucleotide sequence is a barcode, and (iii) the third nucleotide sequence, wherein the third nucleotide sequence is a reverse primer binding site; and (d) inserting the native barcode polynucleotide cassette into the genome of a host cell of the same species as the donor cell in (a); wherein the nucleotide sequences of (i), (ii), and (iii) are native to the host cell.

In some aspects, the donor cell and the host cell are selected from a bacterial cell, a fungal cell, a plant cell, an animal cell, a protozoal cell, and an insect cell. In some aspects, the donor cell and the host cell are each a bacterial cell.

In some aspects, the nucleotide sequence of at least one of (i), (ii), and (iii) is isolated from ribosomal DNA or internal transcribed spacer (ITS) DNA. In some aspects, the nucleotide sequences of at least one of (i), (ii), and (iii) are isolated from 16S rDNA or 18S rDNA.

In some aspects, at least one of the primer binding sites is selected from 8F, 27F, CCF, 357F, 515F, 533F, 16S.1100.F16, 804F, 1237F, 338R, 519R, CDR, 806R, 907R, 1100R, 1391R, 1392R, 1492R(l), and 1492R(s).

In some aspects, the first nucleotide sequence in the genome of the donor cell and the third nucleotide sequence in the genome of the donor cell occur in the same orientation as one another. In some aspects, at least one of (i), (ii), and (iii) do not naturally occur immediately adjacent to one another in the genome of the host cell. In some aspects, the barcode consists of fewer than 100 nucleotides. In some aspects, one or more of the primer binding sites consist of fewer than 30 nucleotides. In some aspects, the nucleotide sequences of the primer binding site and the barcode collectively consist of fewer than 160 nucleotides.

In some aspects, the barcode comprises a constant barcode region and a variable barcode region.

In some aspects, the insertion of the cassette into the genome of the host cell introduces one or more stop codons in either orientation in the host cell. In some aspects, the cassette is inserted in the genome of the host cell between two coding regions separated by a terminator region. In some aspects, the cassette is inserted between the terminator region and one of the two coding regions.

In some aspects, the host cell in (d) is of the same strain as the donor cell in (a).

In some aspects, the native barcode polynucleotide cassette is inserted into the genome of two or more host cells at loci that are set distance away from an origin of replication in the genome of the host cell. In some aspects, the set distance from the origin of replication is similar or identical in each of the two or more host cells. The origin of replication can be in the genome of the host cell or in a self-replicating extrachromosomal genetic entity (e.g., episome or plasmid).

In some aspects, the bacterial host cell is transgenic. In some aspects, the bacterial host cell is a non-intergeneric remodeled bacterium. In some aspects, the non-intergeneric remodeled bacterial host cell is, or is derived from, a bacterium selected from Table 1. In some aspects, the non-intergeneric remodeled bacterium comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network, such that the modified bacterium is capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.

In some aspects, the bacterial host cell is capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. In some aspects, the bacterial host cell comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network. In some aspects, the bacterial host cell comprises an introduced control sequence operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network. In some aspects, the bacterial host cell comprises a heterologous promoter operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.

In some aspects, the bacterial host cell comprises at least one genetic variation introduced into a member selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, a gene associated with biosynthesis of a nitrogenase enzyme, and combinations thereof.

In some aspects, the bacterial host cell comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB, decreased adenylyl-removing activity of GlnE; and decreased uridylyl-removing activity of GlnD.

In some aspects, the bacterial host cell comprises a mutated nifL gene comprising a heterologous promoter in said nifL gene. In some aspects, the bacterial host cell comprises a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain. In some aspects, the bacterial host cell comprises a mutated amtB gene that results in the lack of expression of said amtB gene.

In some aspects, the bacterial host cell comprises at least one of: a mutated nifL gene comprising a heterologous promoter in said nifL gene; a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; and combinations thereof. In some aspects, the bacterial host cell comprises a mutated nifL gene comprising a heterologous promoter in said nifL gene and a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.

In some aspects, the bacterial host cell comprises a mutated nifL gene comprising a heterologous promoter in said nifL gene, a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain, and a mutated amtB gene that results in the lack of expression of said amtB gene.

In some aspects, the bacterial host cell is selected from Rahnella aquatilis, Klebsiella variicola, Achromobacter spiritinus, Achromobacter marplatensis, Microbacterium murale, Kluyvera intermedia, Kosakonia pseudosacchari, Enterobacter sp., Azospirillum lipoferum, and Kosakonia sacchari. In some aspects, the bacterial host cell is endophytic, epiphytic, or rhizospheric. In some aspects, the bacterial host cell is selected from: a bacterium deposited as ATCC PTA-126575, a bacterium deposited as ATCC PTA-126576, a bacterium deposited as ATCC PTA-126577, a bacterium deposited as ATCC PTA-126578, a bacterium deposited as ATCC PTA-126579, a bacterium deposited as ATCC PTA-126580, a bacterium deposited as ATCC PTA-126581, a bacterium deposited as ATCC PTA-126582, a bacterium deposited as ATCC PTA-126583, a bacterium deposited as ATCC PTA-126584, a bacterium deposited as ATCC PTA-126585, a bacterium deposited as ATCC PTA-126586, a bacterium deposited as ATCC PTA-126587, a bacterium deposited as ATCC PTA-126588, a bacterium deposited as NCMA 201701001, a bacterium deposited as NCMA 201701002, a bacterium deposited as NCMA 201701003, a bacterium deposited as NCMA 201708004, a bacterium deposited as NCMA 201708003, a bacterium deposited as NCMA 201708002, a bacterium deposited as NCMA 201708001, a bacterium deposited as NCMA 201712001, and a bacterium deposited as NCMA 201712002.

In some aspects, the bacterial host cell comprises a nucleic acid sequence that shares at least about 95% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 177-260 and 296-303. In some aspects, the bacterial host cell comprises a nucleic acid sequence that shares at least about 99% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 177-260 and 296-303. In some aspects, the bacterial host cell comprises a nucleic acid sequence selected from SEQ ID NOs. 177-260 and 296-303.

In some aspects, the disclosure is drawn to a composition comprising, (a) one or more modified cells comprising, in their genome: (i) a naturally occurring first nucleotide sequence, second nucleotide sequence, and third nucleotide sequence, and (ii) a native barcode polynucleotide cassette comprising the first, second, and third nucleotide sequences oriented 5′ to 3′ as: (1) the first nucleotide sequence, wherein the first nucleotide sequence is a forward primer binding site, (2) the second nucleotide sequence, wherein the second nucleotide sequence is a barcode, and (3) the third nucleotide sequence, wherein the third nucleotide sequence is a reverse primer binding site; wherein the first, second, and third nucleotide sequences of (ii) are in a different proximity to one another as compared to the naturally occurring sequences of (i).

In some aspects, the barcode is unique to each different species of the one or more modified cells. In some aspects, the barcode is unique to each different strain of the one or more modified cells. In some aspects, the one or more modified cells comprise a homogenous population of cells. In some aspects, the one or more modified cells comprise a heterogeneous population of cells.

In some aspects, the heterogeneous population of cells comprises two or more different species. In some aspects, the heterogeneous population of cells comprises two or more different strains. In some aspects, the heterogeneous population of cells comprises two or more strains of each species.

In some aspects, the one or more modified cells is selected from a bacterial cell, a fungal cell, a plant cell, an animal cell, a protozoal cell, and an insect cell. In some aspects, the one or more modified cells is a bacterial cell.

In some aspects, the nucleotide sequence of at least one of (1), (2), or (3) is isolated from ribosomal DNA or internal transcribed spacer (ITS) DNA. In some aspects, the nucleotide sequence of at least one of (1), (2), or (3) is 16S rDNA or 18S rDNA. In some aspects, at least one of the primer binding sites is selected from 8F, 27F, CCF, 357F, 515F, 533F, 16S.1100.F16, 804F, 1237F, 338R, 519R, CDR, 806R, 907R, 1100R, 1391R, 1392R, 1492R(l), and 1492R(s)

In some aspects, the naturally occurring first and third nucleotide sequences in the genome of the modified cells occur in the same orientation as one another. In some aspects, at least one of the naturally occurring first, second, or third nucleotide sequences do not occur immediately adjacent to one another in the genome of the modified cell.

In some aspects, the barcode consists of fewer than 100 nucleotides. In some aspects, one or more of the primer binding sites consist of fewer than 30 nucleotides. In some aspects, the nucleotide sequences of the primer binding sites and the barcode collectively consist of fewer than 160 nucleotides.

In some aspects, the barcode comprises a constant barcode region and a variable barcode region. In some aspects, the constant barcode region of the barcode is the same in each of the one or more modified cells. In some aspects, the variable barcode region of the barcode is different in each of the one or more modified cells, with only cells of the same strain, species, or other categorical distinction sharing the same variable region.

In some aspects, the cassette possesses one or more stop codons in either orientation in the one or more modified cells.

In some aspects, the cassette is present in the genome of the one or more modified cells between two coding regions separated by a terminator region. In some aspects, the cassette is present between the terminator region and one of the two coding regions.

In some aspects, the cassette is present in the genome of each of the one or more modified cells at a set distance from an origin of replication. In some aspects, the set distance from the origin of replication is similar or identical in each of the two or more cells.

In some aspects, the one or more modified bacteria are transgenic. In some aspects, the one or more modified bacteria are non-intergeneric remodeled bacteria. In some aspects, the one or more modified bacteria comprise a population of transgenic bacteria. In some aspects, the one or more modified bacteria comprise a population of non-intergeneric remodeled bacteria. In some aspects, the non-intergeneric remodeled bacteria comprise, or are derived from, a bacterium selected from Table 1.

In some aspects, the non-intergeneric remodeled bacteria comprise at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network, such that the modified bacterium is capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. In some aspects, the bacterial cell is capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.

In some aspects, the bacterial cell comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network. In some aspects, the bacterial cell comprises an introduced control sequence operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory-network. In some aspects, the bacterial cell comprises a heterologous promoter operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.

In some aspects, the bacterial cell comprises at least one genetic variation introduced into a member selection from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, a gene associated with biosynthesis of a nitrogenase enzyme, and combinations thereof.

In some aspects, the bacterial cell comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; and decreased uridylyl-removing activity of GlnD.

In some aspects, the bacterial cell comprises a mutated nifL gene comprising a heterologous promoter in said nifL gene. In some aspects, the bacteria cell comprises a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain. In some aspects, the bacterial cell comprises a mutated amtB gene that results in the lack of expression of said amtB gene.

In some aspects, the bacterial cell comprises at least one of: a mutated nifL gene comprising a heterologous promoter in said nifL gene; a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; and combinations thereof. In some aspects, the bacterial cell comprises a mutated nifL gene comprising a heterologous promoter in said nifL gene and a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain. In some aspects, the bacterial cell comprises a mutated nifL gene comprising a heterologous promoter in said nifL gene, a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain, and a mutated amtB gene that results in the lack of expression of said amtB gene.

In some aspects, the bacterial cell is selected from Paraburkholderia tropica, Paraburkholderia xenovorans, Herbaspirillum seropedicae, Herbaspirullum frisingense, Pseudomonas protegens, Pseudomonas syringae, Pseudomonas benzenivorans, Metakosakonia massiliensis, Metakosakonia intestinii, Rahnella aquatilis, Klebsiella variicola, Achromobacter spiritinus, Achromobacter marplatensis. Microbacterium murale, Kluyvera intermedia, Kosakonia pseudosacchari, Enterobacter sp., Azospirillum lipoferum, and Kosakonia sacchari. In some aspects, the bacteria cell is endophytic, epiphytic, or rhizospheric. In some aspects, the bacterial cell is selected from: a bacterium deposited as ATCC PTA-126575, a bacterium deposited as ATCC PTA-126576, a bacterium deposited as ATCC PTA-126577, a bacterium deposited as ATCC PTA-126578, a bacterium deposited as ATCC PTA-126579, a bacterium deposited as ATCC PTA-126580, a bacterium deposited as ATCC PTA-126581, a bacterium deposited as ATCC PTA-126582, a bacterium deposited as ATCC PTA-126583, a bacterium deposited as ATCC PTA-126584, a bacterium deposited as ATCC PTA-126585, a bacterium deposited as ATCC PTA-126586, a bacterium deposited as ATCC PTA-126587, a bacterium deposited as ATCC PTA-126588, a bacterium deposited as NCMA 201701001, a bacterium deposited as NCMA 201701002, a bacterium deposited as NCMA 201701003, a bacterium deposited as NCMA 201708004, a bacterium deposited as NCMA 201708003, a bacterium deposited as NCMA 201708002, a bacterium deposited as NCMA 201708001, a bacterium deposited as NCMA 201712001, and a bacterium deposited as NCMA 201712002.

In some aspects, the bacterial cell comprises a nucleic acid sequence that shares at least about 95% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 177-260 and 296-303. In some aspects, the bacterial cell comprises a nucleic acid sequence that shares at least about 99% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 177-260 and 296-303. In some aspects, the bacterial cell comprises a nucleic acid sequence selected from SEQ ID NOs. 177-260 and 296-303.

In some aspects, the disclosure is drawn to a method of screening cells in a sample, the method comprising: (a) inserting a native barcode polynucleotide cassette into the genomes of two or more cells, wherein the cassette comprises nucleotide sequences native to each of the two or more cells, oriented 5′ to 3′ as: (i) a forward primer binding site, (ii) a barcode unique to each cell, species, or strain, and (iii) a reverse primer binding site; (b) applying the two or more cells to a proliferative medium; (c) collecting a sample of the proliferative medium comprising the two or more cells; and (d) determining the abundance of the two or more cells in the sample by analyzing the abundance of the barcodes. In some aspects, step (c) further comprises lysing the two or more cells.

In some aspects, the abundance is relative abundance. In some aspects, the abundance is absolute abundance. In some aspects, the abundance is determined by PCR. In some aspects, the PCR is selected from multiplex-PCR, dPCR, ddPCR, or qPCR. In some aspects, the abundance is determined by generating and utilizing a standard curve. The standard curve can be generated by adding in or spiking in a sample or samples at a known concentration or a range of known concentrations. The sample or samples can be added into nucleic acid isolated from samples processed during any of the methods (e.g., the coco-seq assay) as described herein. The sample spiked in can be a control barcode or barcodes as provided herein.

In some aspects, the proliferative medium is selected from a liquid medium, a solid medium, or a semi-solid medium. In some aspects, the proliferative medium is soil. In some aspects, the soil comprises one or more plants or plant parts.

In some aspects, the one or more plants or plant parts are selected from non-leguminous plants. In some aspects, the one or more non-leguminous plants is corn.

In some aspects, the period of time between steps (c) and (d) is an amount of time sufficient for at least one doubling of the two or more cells. In some aspects, the forward primer binding site and/or the reverse primer binding site are the same in each of the two or more cells. In some aspects, the GC content of the forward primer binding site and/or the reverse primer binding site is at least 40%.

In some aspects, the two or more cells comprise a homogenous population of cells. In some aspects, the two or more cells comprise a heterogeneous population of cells. In some aspects, the heterogeneous population of cells comprises two or more different species. In some aspects, the heterogeneous population of cells comprises two or more different strains. In some aspects, the heterogeneous population of cells comprises two or more strains of each species.

In some aspects, the two or more cells are selected from bacterial cells, fungal cells, plant cells, animal cells, protozoal cells, and insect cells. In some aspects, the two or more cells are bacterial cells

In some aspects, the nucleotide sequence of at least one of (i), (ii), or (iii) is isolated from ribosomal DNA or internal transcribed spacer (ITS) DNA. In some aspects, the nucleotide sequence of at least one of (i), (ii), or (iii) is 16S rDNA or 18S rDNA.

In some aspects, at least one of the primer binding sites is selected from 8F, 27F, CCF, 357F, 515F, 533F, 16S.1100.F16, 804F, 1237F, 338R, 519R, CDR, 806R, 907R, 1100R, 1391R, 1392R, 1492R(l), and 1492R(s).

In some aspects, the barcode consists of fewer than 100 nucleotides. In some aspects, one or more of the primer binding sites consist of fewer than 30 nucleotides. In some aspects, the nucleotide sequences of the primer binding sites and the barcode collectively consist of fewer than 160 nucleotides.

In some aspects, the two or more bacteria are transgenic. In some aspects, the two or more bacteria are non-intergeneric remodeled bacteria. In some aspects, the two or more bacteria comprise a population of transgenic bacteria. In some aspects, the two or more bacteria comprise a population of non-intergeneric remodeled bacteria. In some aspects, the non-intergeneric remodeled bacteria comprise, or are derived from, a bacterium selected from Table 1. In some aspects, the two or more bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.

In some aspects, the barcode comprises a constant barcode region and a variable barcode region. In some aspects, the constant barcode region of the barcode inserted into each of the two or more cells is the same in each of the two or more cells. In some aspects, the variable barcode region of the barcode inserted into each of the two or more cells is different, with only cells of the same strain, species, or other categorical distinction sharing the same variable region.

In some aspects, the insertion of the cassette into the genome of the two or more cells introduces one or more stop codons in either orientation in the two or more cells.

In some aspects, the cassette is present in the genome of the two or more cells between two coding regions separated by a terminator region. In some aspects, the cassette is present between the terminator region and one of the two coding regions.

In some aspects, the cassette is present in the genome of each of the two or more cells at a set distance from an origin of replication. In some aspects, the set distance from the origin of replication is similar or identical in each of the two or more cells. Ensuring the insertion of the native barcode polynucleotide cassettes at a semi-constant distance from origins of replication in each cell or strain can be utilized to minimize any bias introduced in sequencing coverage due to distance from the origin of replication.

In some aspects, the disclosure is drawn to a method of barcoding a population of host cells, the method comprising: (a) obtaining a donor cell; (b) selecting and isolating a first nucleotide sequence in the genome of the donor cell, selecting and isolating a second nucleotide sequence in the genome of the donor cell, and selecting and isolating a third nucleotide sequence in the genome of the donor cell; (c) creating a native barcode polynucleotide cassette comprising the first, second, and third nucleotide sequences oriented 5′ to 3′ as: (i) the first nucleotide sequence, wherein the first nucleotide sequence is a forward primer binding site, (ii) the second nucleotide sequence, wherein the second nucleotide sequence is a barcode, and (iii) the third nucleotide sequence, wherein the third nucleotide sequence is a reverse primer binding site; (d) inserting the native barcode polynucleotide cassette into the genome of a host cell of the same genus as the donor cell in (a); and (e) repeating (d) one or more times with a different host cell of the same genus each time; wherein the nucleotide sequences of (i), (ii), and (iii) are native to the host cell, thereby barcoding a population of host cells.

In some aspects, the population of host cells share a common species. In some aspects, each of the different host cells of (e) is a different strain. In some aspects, each of the different host cells of (e) is a different species.

In some aspects, the barcode comprises a constant barcode region and a variable barcode region. In some aspects, the constant barcode region of the barcode inserted into each host cell of the population of host cells is the same in each of the host cells. In some aspects, the variable barcode region of the barcode inserted into each host cell of the population of host cells is different, with only host cells of the same strain, species, or other categorical distinction sharing the same variable region. In some aspects, the insertion of the cassette into the genome of the host cell introduces one or more stop codons in either orientation in the host cell. In some aspects, the insertion of the cassette into the genome of each cell in the population is between two coding regions separated by a terminator region. In some aspects, the insertion of the cassette is between the terminator region and one of the two coding regions. In some aspects, the insertion of the cassette into the genome of each cell in the population is at a set distance from an origin of replication in each cell. The set distance can be any distance from the origin of replication, provided that the barcode(s) is consistently incorporated at the same or similar distance from the origin of replication in each cell of the population in which a barcode is incorporated.

In some aspects, the disclosure is drawn to a composition comprising: (a) a population of modified cells comprising, in their genome: (i) a naturally occurring first nucleotide sequence, second nucleotide sequence, and third nucleotide sequence, and (ii) a native barcode polynucleotide cassette comprising the first, second, and third nucleotide sequences oriented 5′ to 3′ as: (1) the first nucleotide sequence, wherein the first nucleotide sequence is a forward primer binding site, (2) the second nucleotide sequence, wherein the second nucleotide sequence is a barcode, and (3) the third nucleotide sequence, wherein the third nucleotide sequence is a reverse primer binding site; wherein the first, second, and third nucleotide sequences of (ii) are in a different proximity to one another as compared to the naturally occurring sequences of (i).

In some aspects, the population of modified cells share a common species. In some aspects, the population of modified cells comprises more than one species. In some aspects, the population of modified cells comprises more than one strain of each species.

In some aspects, the barcode comprises a constant barcode region and a variable barcode region. In some aspects, the constant barcode region is the same in each of the modified cells. In some aspects, the variable barcode region in the population of modified cells not conserved, with only modified cells of the same strain, species, or other categorical distinction sharing the same variable region. In some aspects, insertion of the cassette into the genome of the population of modified cells introduces one or more stop codons in either orientation in the modified cells.

In some aspects, the cassette is present in the genome of the host cell between two coding regions separated by a terminator region. In some aspects, the cassette is present between the terminator region and one of the two coding regions.

In some aspects, the cassette is present in the genome of the host cell at a set distance from an origin of replication. In some aspects, the set distance from the origin of replication is similar or identical in each modified cell of the population of modified cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts an overview of the guided microbial remodeling process, in accordance with embodiments.

FIG. 1B depicts an expanded view of the measurement of microbiome composition as shown in FIG. 1A.

FIG. 1C depicts a problematic “traditional bioprospecting” approach, which has several drawbacks compared to the taught guided microbial remodeling (GMR) platform.

FIG. 1D depicts a problematic “field-first approach to bioprospecting” system, which has several drawbacks compared to the taught guided microbial remodeling (GMR) platform.

FIG. 1E depicts the time period in the corn growth cycle, at which nitrogen is needed most by the plant.

FIG. 1F depicts an overview of a field development process for a remodeled microbe.

FIG. 1G depicts an overview of a guided microbial remodeling platform embodiment.

FIG. 1H depicts an overview of a computationally-guided microbial remodeling platform.

FIG. 1I depicts the use of field data combined with modeling in aspects of the guided microbial remodeling platform.

FIG. 1J depicts 5 properties that can be possessed by remodeled microbes of the present disclosure.

FIG. 1K depicts a schematic of a remodeling approach for a microbe, PBC6.1.

FIG. 1L depicts decoupled nifA expression from endogenous nitrogen regulation in remodeled microbes.

FIG. 1M depicts improved assimilation and excretion of fixed nitrogen by remodeled microbes.

FIG. 1N depicts corn yield improvement attributable to remodeled microbes.

FIG. 1O illustrates the inefficiency of current nitrogen delivery systems, which result in under fertilized fields, over fertilized fields, and environmentally deleterious nitrogen runoff

FIG. 2A illustrates a current plant colonization assays utilized in the GMR process, which is based upon absolute colonization. The described assays and processes are not limited to cells that have been produced as a result of the GMR process.

FIG. 2B depicts hypothetical plant counts that are required to assess the colonization ability of 19 strains using different approaches.

FIG. 3A-FIG. 3D illustrate a workflow of the taught relative colonization assays of Example 2 that are enabled by the utilization of natural microbial barcodes. These relative colonization assays drastically reduce the number of plants required for a colonization assay. FIG. 3A illustrates the selection of naturally occurring conserved regions in a bacterial genome for use as universal primers. FIG. 3B illustrates the selection of naturally occurring sequences from bacterial strains to serve as barcode sequences. FIG. 3C illustrates the cassette comprising the primer binding sites and the barcode. FIG. 3D illustrates the general workflow in planta for evaluation of competitive colonization of the bacteria.

FIG. 4 illustrates optimization of primer binding site pairs in a PCR of pooled templates from genomic DNA of a variety of wildtype bacterial strains. Ideally, wildtype strain templates will produce no product, as observed in lane 4.

FIG. 5 illustrates barcode skewing due to bidirectional replication in a microbial (i.e., E. coli) chromosome.

FIG. 6A illustrates potential insertion sites for native barcode polynucleotide sequences in the genome of a microbe, while FIG. 6B shows the location of a native barcode polynucleotide sequence in the genome of a microbe in relation to the two coding sequences and the terminator sequence located there between.

FIG. 7 illustrates the lower colonization phenotype of the 137-1036 strain vs. the parental 137 strain as reflected by the number of copies of the strains' genomes by qPCR. Each strain represents a single treatment of 12 plants.

FIG. 8 illustrates the differential colonization of 137 strains containing native barcodes (i.e., barcode 1, 2 and 7) as reflected by differences in relative abundance of the different barcode labelled strains growing on plants inoculated with an inoculum containing each of the native barcodes (i.e., barcodes 1, 2 and 7) in a 100:10:1 ratio, respectively.

FIG. 9 illustrates the lower colonization phenotype of the 137-1036 strain vs. the parental 137 strain as reflected by differences in relative abundance of different barcode labelled strains growing on plants inoculated with a pool of the different barcode labelled strains.

FIG. 10 illustrates reads counts determined for known copy numbers of control DNA and test DNA using the amplification and sequencing method described in Examples 2 and 3 and throughout this disclosure. The control DNA and the test DNA both possess the same universal primer binding sites that flank barcodes sequences that are unique to either the control DNA or the test DNA. The unique barcode sequence present in the control DNA was based on sequences from the sea otter genome, while the unique barcode sequence present in the test DNA was chosen based on sequences from the 137 parental strain as described in Example 2. The control DNA was spiked in at different copy numbers that spanned several orders of magnitude (i.e., 10⁶, 10³ and 10⁰ in test 1 or 10⁷, 10⁴, and 10¹ in test 2), while the test DNA was added at a known level, 10⁵ copies for both tests 1 and 2.

FIG. 11 illustrates total abundance (cells per gram of fresh weight (gfw)) for strains 137, 137-1036, 137-3933, 137-3944 and 137-2285 as determined using the amplification and sequencing method described in Example 4 and throughout this disclosure. Each of the strains contain native polynucleotide barcode sequences that contain the same universal primer binding sites that flank barcodes sequences that are unique to each of the strains as described in Example 4. As can be seen, the Coco-seq assay described in Example 4 revealed that each of the barcoded strains possess a weaker colonization capacity than the parental 137 strain with strain 137-2285 possessing the weakest colonization capacity.

DETAILED DESCRIPTION OF THE DISCLOSURE

While various embodiments of the disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.

Increased fertilizer utilization brings with it environmental concerns and is also likely not possible for many economically stressed regions of the globe. Furthermore, many industry players in the microbial arena are focused on creating intergeneric microbes. However, there is a heavy regulatory burden placed on engineered microbes that are characterized/classified as intergeneric. These intergeneric microbes face not only a higher regulatory burden, which makes widespread adoption and implementation difficult, but they also face a great deal of public perception scrutiny.

Currently, there are no engineered microbes on the market that are non-intergeneric and that are capable of increasing nitrogen fixation in non-leguminous crops. This dearth of such a microbe is a missing element in helping to usher in a truly environmentally friendly and more sustainable 21^(st) century agricultural system.

An issue that arises in the development of such an organism is the ability to identify candidates that are not only able to fix nitrogen but also able to colonize the roots of non-leguminous crops. When screening candidates, organisms that are highly efficient at nitrogen fixation but are less efficient at colonization, may be hard to detect in mixed samples. The present disclosure solves the aforementioned problems and provides a method for increasing the ability to detect such organisms. The taught methods and non-intergeneric microbes identified thereby will serve to help 21^(st) century farmers become less dependent upon utilizing ever increasing amounts of exogenous nitrogen fertilizer.

Definitions

The use of the terms “a” and “an” and “the” and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10-15 is disclosed, then 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

“Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner according to base complementarity. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the enzymatic cleavage of a polynucleotide by an endonuclease. A second sequence that is complementary to a first sequence is referred to as the “complement” of the first sequence. The term “hybridizable” as applied to a polynucleotide refers to the ability of the polynucleotide to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues in a hybridization reaction.

As used herein, “biofilm” or “mature biofilm” refers to associated and/or accumulated and/or aggregated microbial cells, their products (e.g. exopolymeric substances) and inorganic particles adherent to a living or inert surface.

“Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. Sequence identity, such as for the purpose of assessing percent complementarity, may be measured by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see e.g. the EMBOSS Needle aligner available at www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html, optionally with default settings), the BLAST algorithm (see e.g. the BLAST alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionally with default settings), or the Smith-Waterman algorithm (see e.g. the EMBOSS Water aligner available at www.ebi.ac.uk/Tools/psa/emboss_water/nucleotide.html, optionally with default settings). Optimal alignment may be assessed using any suitable parameters of a chosen algorithm, including default parameters.

In general, “stringent conditions” for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with a target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter “Overview of principles of hybridization and the strategy of nucleic acid probe assay”, Elsevier, N.Y.

As used herein, “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein the term “amino acid” includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.

As used herein, the term “about” is used synonymously with the term “approximately.” Illustratively, the use of the term “about” with regard to an amount indicates that values slightly outside the cited values, e.g., plus or minus 0.1% to 10%.

The term “biologically pure culture” or “substantially pure culture” refers to a culture of a bacterial species described herein containing no other bacterial species in quantities sufficient to interfere with the replication of the culture or be detected by normal bacteriological techniques.

“Plant productivity” refers generally to any aspect of growth or development of a plant that is a reason for which the plant is grown. For food crops, such as grains or vegetables, “plant productivity” can refer to the yield of grain or fruit harvested from a particular crop. As used herein, improved plant productivity refers broadly to improvements in yield of grain, fruit, flowers, or other plant parts harvested for various purposes, improvements in growth of plant parts, including stems, leaves and roots, promotion of plant growth, maintenance of high chlorophyll content in leaves, increasing fruit or seed numbers, increasing fruit or seed unit weight, reducing NO₂ emission due to reduced nitrogen fertilizer usage and similar improvements of the growth and development of plants.

Microbes in and around food crops can influence the traits of those crops. Plant traits that may be influenced by microbes include, yield (e.g., grain production, biomass generation, fruit development, flower set), nutrition (e.g., nitrogen, phosphorus, potassium, iron, micronutrient acquisition); abiotic stress management (e.g., drought tolerance, salt tolerance, heat tolerance); and biotic stress management (e.g., pest, weeds, insects, fungi, and bacteria). Strategies for altering crop traits include: increasing key metabolite concentrations; changing temporal dynamics of microbe influence on key metabolites; linking microbial metabolite production/degradation to new environmental cues; reducing negative metabolites; and improving the balance of metabolites or underlying proteins.

As used herein, a “control sequence” refers to an operator, promoter, silencer, or terminator.

As used herein, “in planta” may refer to in the plant, on the plant or intimately associated with the plant, depending upon context of usage (e.g. endophytic, epiphytic, or rhizospheric associations). The plant may comprise plant parts, tissue, leaves, roots, root hairs, rhizomes, stems, seed, ovules, pollen, flowers, fruit etc.

In some embodiments, native or endogenous control sequences of genes of the present disclosure are replaced with one or more intrageneric control sequences.

As used herein, “introduced” refers to the introduction by means of modern biotechnology, and not a naturally occurring introduction.

In some embodiments, the bacteria of the present disclosure have been modified such that they are not naturally occurring bacteria.

In some embodiments, the bacteria of the present disclosure are present in the plant in an amount of at least 10³ cfu, 10⁴ cfu, 10⁵ cfu, 10⁶ cfu, 10⁷ cfu, 10⁸ cfu, 10⁹ cfu, 10¹⁰ cfu, 10¹¹ cfu, or 10¹² cfu per gram of fresh or dry weight of the plant. In some embodiments, the bacteria of the present disclosure are present in the plant in an amount of at least about 10³ cfu, about 10⁴ cfu, about 10⁵ cfu, about 10⁶ cfu, about 10⁷ cfu, about 10⁸ cfu, about 10⁹ cfu, about 10¹⁰ cfu, about 10¹¹ cfu, or about 10¹² cfu per gram of fresh or dry weight of the plant. In some embodiments, the bacteria of the present disclosure are present in the plant in an amount of at least 10³ to 10⁹, 10³ to 10⁷, 10³ to 10⁵, 10⁵ to 10⁹, 10⁵ to 10⁷, 10⁶ to 10¹⁰, 10⁶ to 10⁷ cfu per gram of fresh or dry weight of the plant.

Fertilizers and exogenous nitrogen of the present disclosure may comprise the following nitrogen-containing molecules: ammonium, nitrate, nitrite, ammonia, glutamine, etc. Nitrogen sources of the present disclosure may include anhydrous ammonia, ammonia sulfate, urea, diammonium phosphate, urea-form, monoammonium phosphate, ammonium nitrate, nitrogen solutions, calcium nitrate, potassium nitrate, sodium nitrate, etc.

As used herein, “exogenous nitrogen” refers to non-atmospheric nitrogen readily available in the soil, field, or growth medium that is present under non-nitrogen limiting conditions, including ammonia, ammonium, nitrate, nitrite, urea, uric acid, ammonium acids, etc.

As used herein, “non-nitrogen limiting conditions” refers to non-atmospheric nitrogen available in the soil, field or media at concentrations greater than about 4 mM nitrogen, as disclosed by Kant et al. (2010. J. Exp. Biol. 62(4): 1499-1509), which is incorporated herein by reference.

As used herein, an “intergeneric microorganism” is a microorganism that is formed by the deliberate combination of genetic material originally isolated from organisms of different taxonomic genera. An “intergeneric mutant” can be used interchangeably with “intergeneric microorganism”. An exemplary “intergeneric microorganism” includes a microorganism containing a mobile genetic element which was first identified in a microorganism in a genus different from the recipient microorganism. Further explanation can be found, inter alia, in 40 C.F.R. § 725.3.

In aspects, microbes taught herein are “non-intergeneric,” which means that the microbes are not intergeneric.

As used herein, an “intrageneric microorganism” is a microorganism that is formed by the deliberate combination of genetic material originally isolated from organisms of the same taxonomic genera. An “intrageneric mutant” can be used interchangeably with “intrageneric microorganism”.

As used herein, “introduced genetic material” means genetic material that is added to, and remains as a component of, the genome of the recipient.

As used herein, in the context of non-intergeneric microorganisms, the term “remodeled” is used synonymously with the term “engineered”. Consequently, a “non-intergeneric remodeled microorganism” has a synonymous meaning to “non-intergeneric engineered microorganism,” and will be utilized interchangeably. Further, the disclosure may refer to an “engineered strain” or “engineered derivative” or “engineered non-intergeneric microbe,” these terms are used synonymously with “remodeled strain” or “remodeled derivative” or “remodeled non-intergeneric microbe.”

In some embodiments, the nitrogen fixation and assimilation genetic regulatory network comprises polynucleotides encoding genes and non-coding sequences that direct, modulate, and/or regulate microbial nitrogen fixation and/or assimilation and can comprise polynucleotide sequences of the nif cluster (e.g., nifA, nifB, nifC, . . . nifZ), polynucleotides encoding nitrogen regulatory protein C, polynucleotides encoding nitrogen regulatory protein B, polynucleotide sequences of the gln cluster (e.g. glnA and glnD), draT, and ammonia transporters/permeases. In some cases, the Nif cluster may comprise NifB, NifH, NifD, NifK, NifE, NifN, NifX, hesa, and NifV. In some cases, the Nif cluster may comprise a subset of NifB, NifH, NifD, NifK, NifE, NifN, NifX, hesa, and NifV.

In some embodiments, fertilizer of the present disclosure comprises at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% nitrogen by weight.

In some embodiments, fertilizer of the present disclosure comprises at least about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% nitrogen by weight.

In some embodiments, fertilizer of the present disclosure comprises about 5% to 50%, about 5% to 75%, about 10% to 50%, about 10% to 75%, about 15% to 50%, about 15% to 75%, about 20% to 50%, about 20% to 75%, about 25% to 50%, about 25% to 75%, about 30% to 50%, about 30% to 75%, about 35% to 50%, about 35% to 75%, about 40% to 50%, about 40% to 75%, about 45% to 50%, about 45% to 75%, or about 50% to 75% nitrogen by weight.

In some embodiments, the increase of nitrogen fixation and/or the production of 1% or more of the nitrogen in the plant are measured relative to control plants, which have not been exposed to the bacteria of the present disclosure. All increases or decreases in bacteria are measured relative to control bacteria. All increases or decreases in plants are measured relative to control plants.

As used herein, a “constitutive promoter” is a promoter, which is active under most conditions and/or during most development stages. There are several advantages to using constitutive promoters in expression vectors used in biotechnology, such as: high level of production of proteins used to select transgenic cells or organisms; high level of expression of reporter proteins or scorable markers, allowing easy detection and quantification; high level of production of a transcription factor that is part of a regulatory transcription system; production of compounds that requires ubiquitous activity in the organism; and production of compounds that are required during all stages of development. Non-limiting exemplary constitutive promoters include, CaMV 35S promoter, opine promoters, ubiquitin promoter, alcohol dehydrogenase promoter, etc.

As used herein, a “non-constitutive promoter” is a promoter which is active under certain conditions, in certain types of cells, and/or during certain development stages. For example, tissue specific, tissue preferred, cell type specific, cell type preferred, inducible promoters, and promoters under development control are non-constitutive promoters. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues.

As used herein, “inducible” or “repressible” promoter is a promoter which is under chemical or environmental factors control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, certain chemicals, the presence of light, acidic or basic conditions, etc.

As used herein, a “tissue specific” promoter is a promoter that initiates transcription only in certain tissues. Unlike constitutive expression of genes, tissue-specific expression is the result of several interacting levels of gene regulation. As such, in the art sometimes it is preferable to use promoters from homologous or closely related species to achieve efficient and reliable expression of transgenes in particular tissues. This is one of the main reasons for the large amount of tissue-specific promoters isolated from particular tissues found in both scientific and patent literature.

As used herein, the term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation. In another example, the complementary RNA regions of the disclosure can be operably linked, either directly or indirectly, 5′ to the target mRNA, or 3′ to the target mRNA, or within the target mRNA, or a first complementary region is 5′ and its complement is 3′ to the target mRNA.

In aspects, “applying to the plant a plurality of non-intergeneric bacteria,” includes any means by which the plant (including plant parts such as a seed, root, stem, tissue, etc.) is made to come into contact (i.e. exposed) with said bacteria at any stage of the plant's life cycle. Consequently, “applying to the plant a plurality of non-intergeneric bacteria,” includes any of the following means of exposing the plant (including plant parts such as a seed, root, stem, tissue, etc.) to said bacteria: spraying onto plant, dripping onto plant, applying as a seed coat, applying to a field that will then be planted with seed, applying to a field already planted with seed, applying to a field with adult plants, etc.

As used herein “MRTN” is an acronym for maximum return to nitrogen and is utilized as an experimental treatment in the Examples. MRTN was developed by Iowa State University and information can be found at: cnrc.agron.iastate.edu/. The MRTN is the nitrogen rate where the economic net return to nitrogen application is maximized. The approach to calculating the MRTN is a regional approach for developing corn nitrogen rate guidelines in individual states. The nitrogen rate trial data was evaluated for Illinois, Iowa, Michigan, Minnesota, Ohio, and Wisconsin where an adequate number of research trials were available for corn plantings following soybean and corn plantings following corn. The trials were conducted with spring, side dress, or split preplant/side dress applied nitrogen, and sites were not irrigated except for those that were indicated for irrigated sands in Wisconsin. MRTN was developed by Iowa State University due to apparent differences in methods for determining suggested nitrogen rates required for corn production, misperceptions pertaining to nitrogen rate guidelines, and concerns about application rates. By calculating the MRTN, practitioners can determine the following: (1) the nitrogen rate where the economic net return to nitrogen application is maximized, (2) the economic optimum nitrogen rate, which is the point where the last increment of nitrogen returns a yield increase large enough to pay for the additional nitrogen, (3) the value of corn grain increase attributed to nitrogen application, and the maximum yield, which is the yield where application of more nitrogen does not result in a corn yield increase. Thus the MRTN calculations provide practitioners with the means to maximize corn crops in different regions while maximizing financial gains from nitrogen applications.

The term mmol is an abbreviation for millimole, which is a thousandth (10⁻³) of a mole, abbreviated herein as mol.

As used herein the terms “microorganism” or “microbe” should be taken broadly. These terms, used interchangeably, include but are not limited to, the two prokaryotic domains. Bacteria and Archaea. The term may also encompass eukaryotic fungi and protists.

The term “microbial consortia” or “microbial consortium” refers to a subset of a microbial community of individual microbial species, or strains of a species, which can be described as carrying out a common function, or can be described as participating in, or leading to, or correlating with, a recognizable parameter, such as a phenotypic trait of interest.

The term “microbial community” means a group of microbes comprising two or more species or strains. Unlike microbial consortia, a microbial community does not have to be carrying out a common function, or does not have to be participating in, or leading to, or correlating with, a recognizable parameter, such as a phenotypic trait of interest.

As used herein, “isolate,” “isolated,” “isolated microbe,” and like terms, are intended to mean that the one or more microorganisms has been separated from at least one of the materials with which it is associated in a particular environment (for example soil, water, plant tissue, etc). Thus, an “isolated microbe” does not exist in its naturally occurring environment; rather, it is through the various techniques described herein that the microbe has been removed from its natural setting and placed into a non-naturally occurring state of existence. Thus, the isolated strain or isolated microbe may exist as, for example, a biologically pure culture, or as spores (or other forms of the strain). In aspects, the isolated microbe may be in association with an acceptable carrier, which may be an agriculturally acceptable carrier.

In certain aspects of the disclosure, the isolated microbes exist as “isolated and biologically pure cultures.” It will be appreciated by one of skill in the art that an isolated and biologically pure culture of a particular microbe, denotes that said culture is substantially free of other living organisms and contains only the individual microbe in question. The culture can contain varying concentrations of said microbe. The present disclosure notes that isolated and biologically pure microbes often “necessarily differ from less pure or impure materials.” See, e.g. In re Bergstrom, 427 F.2d 1394, (CCPA 1970) discussing purified prostaglandins), see also, In re Bergy, 596 F.2d 952 (CCPA 1979)(discussing purified microbes), see also, Parke-Davis & Co. v. H. K. Mulford & Co., 189 F. 95 (S.D.N.Y. 1911) (Learned Hand discussing purified adrenaline), aff'd in part rev'd in part, 1% F. 4% (2d Cir. 1912), each of which are incorporated herein by reference. Furthermore, in some aspects, the disclosure provides for certain quantitative measures of the concentration, or purity limitations, that must be found within an isolated and biologically pure microbial culture. The presence of these purity values, in certain embodiments, is a further attribute that distinguishes the presently disclosed microbes from those microbes existing in a natural state. See, e.g., Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4th Cir. 1958) (discussing purity limitations for vitamin B12 produced by microbes), incorporated herein by reference.

As used herein, “individual isolates” should be taken to mean a composition, or culture, comprising a predominance of a single genera, species, or strain, of microorganism, following separation from one or more other microorganisms.

Microbes of the present disclosure may include spores and/or vegetative cells. In some embodiments, microbes of the present disclosure include microbes in a viable but non-culturable (VBNC) state. As used herein, “spore” or “spores” refer to structures produced by bacteria and fungi that are adapted for survival and dispersal. Spores are generally characterized as dormant structures; however, spores are capable of differentiation through the process of germination. Germination is the differentiation of spores into vegetative cells that are capable of metabolic activity, growth, and reproduction. The germination of a single spore results in a single fungal or bacterial vegetative cell. Fungal spores are units of asexual reproduction, and in some cases are necessary structures in fungal life cycles. Bacterial spores are structures for surviving conditions that may ordinarily be nonconductive to the survival or growth of vegetative cells.

As used herein, “microbial composition” refers to a composition comprising one or more microbes of the present disclosure. In some embodiments, a microbial composition is administered to plants (including various plant parts) and/or in agricultural fields.

As used herein, “carrier,” “acceptable carrier,” or “agriculturally acceptable carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the microbe can be administered, which does not detrimentally effect the microbe.

Regulation of Nitrogen Fixation

In some cases, nitrogen fixation pathway may act as a target for genetic engineering and optimization. One trait that may be targeted for regulation by the methods described herein is nitrogen fixation. Nitrogen fertilizer is the largest operational expense on a farm and the biggest driver of higher yields in row crops like corn and wheat. Described herein are microbial products that can deliver renewable forms of nitrogen in non-leguminous crops. While some endophytes have the genetics necessary for fixing nitrogen in pure culture, the fundamental technical challenge is that wild-type endophytes of cereals and grasses stop fixing nitrogen in fertilized fields. The application of chemical fertilizers and residual nitrogen levels in field soils signal the microbe to shut down the biochemical pathway for nitrogen fixation.

Changes to the transcriptional and post-translational levels of components of the nitrogen fixation regulatory network may be beneficial to the development of a microbe capable of fixing and transferring nitrogen to corn in the presence of fertilizer. To that end, described herein is Host-Microbe Evolution (HoME) technology to precisely evolve regulatory networks and elicit novel phenotypes. Also described herein are unique, proprietary libraries of nitrogen-fixing endophytes isolated from corn, paired with extensive omics data surrounding the interaction of microbes and host plant under different environmental conditions like nitrogen stress and excess. In some embodiments, this technology enables precision evolution of the genetic regulatory network of endophytes to produce microbes that actively fix nitrogen even in the presence of fertilizer in the field. Also described herein are evaluations of the technical potential of evolving microbes that colonize corn root tissues and produce nitrogen for fertilized plants and evaluations of the compatibility of endophytes with standard formulation practices and diverse soils to determine feasibility of integrating the microbes into modern nitrogen management strategies.

In order to utilize elemental nitrogen (N) for chemical synthesis, life forms combine nitrogen gas (N₂) available in the atmosphere with hydrogen in a process known as nitrogen fixation. Because of the energy-intensive nature of biological nitrogen fixation, diazotrophs (bacteria and archaea that fix atmospheric nitrogen gas) have evolved sophisticated and tight regulation of the nif gene cluster in response to environmental oxygen and available nitrogen. Nif genes encode enzymes involved in nitrogen fixation (such as the nitrogenase complex) and proteins that regulate nitrogen fixation. Shamseldin (2013. Global J. Biotechnol Biochem. 8(4) 84-94) discloses detailed descriptions of nif genes and their products, and is incorporated herein by reference. Described herein are methods of producing a plant with an improved trait comprising isolating bacteria from a first plant, introducing a genetic variation into a gene of the isolated bacteria to increase nitrogen fixation, exposing a second plant to the variant bacteria, isolating bacteria from the second plant having an improved trait relative to the first plant, and repeating the steps with bacteria isolated from the second plant.

In Proteobacteria, regulation of nitrogen fixation centers around the ow-dependent enhancer-binding protein NifA, the positive transcriptional regulator of the nif cluster. Intracellular levels of active NifA are controlled by two key factors, transcription of the nifLA operon, and inhibition of NifA activity by protein-protein interaction with NifL. Both of these processes are responsive to intracellular glutamine levels via the PII protein signaling cascade. This cascade is mediated by GlnD, which directly senses glutamine and catalyzes the uridylylation or deuridylylation of two PII regulatory proteins—GlnB and GlnK—in response the absence or presence, respectively, of bound glutamine. Under conditions of nitrogen excess, unmodified GlnB signals the deactivation of the nifLA promoter. However, under conditions of nitrogen limitation, GlnB is post-translationally modified, which inhibits its activity and leads to transcription of the nifLA operon. In this way, nifLA transcription is tightly controlled in response to environmental nitrogen via the PII protein signaling cascade. On the post-translational level of NifA regulation, GlnK inhibits the NifL/NifA interaction in a matter dependent on the overall level of free GlnK within the cell.

NifA is transcribed from the nifLA operon, whose promoter is activated by phosphorylated NtrC, another σ₅₄-dependent regulator. The phosphorylation state of NtrC is mediated by the histidine kinase NtrB, which interacts with deuridylylated GlnB but not uridylylated GlnB. Under conditions of nitrogen excess, a high intracellular level of glutamine leads to deuridylylation of GlnB, which then interacts with NtrB to deactivate its phosphorylation activity and activate its phosphatase activity, resulting in dephosphorylation of NtrC and the deactivation of the nifLA promoter. However, under conditions of nitrogen limitation, a low level of intracellular glutamine results in uridylylation of GlnB, which inhibits its interaction with NtrB and allows the phosphorylation of NtrC and transcription of the nifLA operon. In this way, nifLA expression is tightly controlled in response to environmental nitrogen via the PII protein signaling cascade, nif A, ntrB, ntrC, and glnB, are all genes that can be mutated in the methods described herein. These processes may also be responsive to intracellular or extracellular levels of ammonia, urea or nitrates.

The activity of NifA is also regulated post-translationally in response to environmental nitrogen, most typically through NifL-mediated inhibition of NifA activity. In general, the interaction of NifL and NifA is influenced by the PII protein signaling cascade via GlnK, although the nature of the interactions between GlnK and NifL/NifA varies significantly between diazotrophs. In Klebsiella pneumoniae, both forms of GlnK inhibit the NifL/NifA interaction, and the interaction between GlnK and NifL/NifA is determined by the overall level of free GlnK within the cell. Under nitrogen-excess conditions, deuridylylated GlnK interacts with the ammonium transporter AmtB, which serves to both block ammonium uptake by AmtB and sequester GlnK to the membrane, allowing inhibition of NifA by NifL. On the other hand, in Azotobacter vinelandii, interaction with deuridylylated GlnK is required for the NifL/NifA interaction and NifA inhibition, while uridylylation of GlnK inhibits its interaction with NifL. In diazotrophs lacking the nifL gene, there is evidence that NifA activity is inhibited directly by interaction with the deuridylylated forms of both GlnK and GlnB under nitrogen-excess conditions. In some bacteria the Nif cluster may be regulated by glnR, and further in some cases this may comprise negative regulation. Regardless of the mechanism, post-translational inhibition of NifA is an important regulator of the nif cluster in most known diazotrophs. Additionally, nifL, amtB, glnK, and glnR are genes that can be mutated in the methods described herein.

In addition to regulating the transcription of the nif gene cluster, many diazotrophs have evolved a mechanism for the direct post-translational modification and inhibition of the nitrogenase enzyme itself, known as nitrogenase shutoff. This is mediated by ADP-ribosylation of the Fe protein (NifH) under nitrogen-excess conditions, which disrupts its interaction with the MoFe protein complex (NifDK) and abolishes nitrogenase activity. DraT catalyzes the ADP-ribosylation of the Fe protein and shutoff of nitrogenase, while DraG catalyzes the removal of ADP-ribose and reactivation of nitrogenase. As with nifLA transcription and NifA inhibition, nitrogenase shutoff is also regulated via the PII protein signaling cascade. Under nitrogen-excess conditions, deuridylylated GlnB interacts with and activates DraT, while deuridylylated GlnK interacts with both DraG and AmtB to form a complex, sequestering DraG to the membrane. Under nitrogen-limiting conditions, the uridylylated forms of GlnB and GlnK do not interact with DraT and DraG, respectively, leading to the inactivation of DraT and the diffusion of DraG to the Fe protein, where it removes the ADP-ribose and activates nitrogenase. The methods described herein also contemplate introducing genetic variation into the nifH, nifD, nifK, and draT genes.

Although some endophytes have the ability to fix nitrogen in vitro, often the genetics are silenced in the field by high levels of exogenous chemical fertilizers. One can decouple the sensing of exogenous nitrogen from expression of the nitrogenase enzyme to facilitate field-based nitrogen fixation. Improving the integral of nitrogenase activity across time further serves to augment the production of nitrogen for utilization by the crop. Specific targets for genetic variation to facilitate field-based nitrogen fixation using the methods described herein include one or more genes selected from the group consisting of nifA, nifL, ntrB, ntrC, glnA, glnB, glnK, draT, amtB, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, and nifQ.

An additional target for genetic variation to facilitate field-based nitrogen fixation using the methods described herein is the NifA protein. The NifA protein is typically the activator for expression of nitrogen fixation genes. Increasing the production of NifA (either constitutively or during high ammonia condition) circumvents the native ammonia-sensing pathway. In addition, reducing the production of NifL proteins, a known inhibitor of NifA, also leads to an increased level of freely active NifA. In addition, increasing the transcription level of the nifAL operon (either constitutively or during high ammonia condition) also leads to an overall higher level of NifA proteins. Elevated level of nifAL expression is achieved by altering the promoter itself or by reducing the expression of NtrB (part of ntrB and ntrC signaling cascade that originally would result in the shutoff of nifAL operon during high nitrogen condition). High level of NifA achieved by these or any other methods described herein increases the nitrogen fixation activity of the endophytes.

Another target for genetic variation to facilitate field-based nitrogen fixation using the methods described herein is the GlnD/GlnB/GlnK PII signaling cascade. The intracellular glutamine level is sensed through the GlnD/GlnB/GlnK PII signaling cascade. Active site mutations in GlnD that abolish the uridylyl-removing activity of GlnD disrupt the nitrogen-sensing cascade. In addition, reduction of the GlnB concentration short circuits the glutamine-sensing cascade. These mutations “trick” the cells into perceiving a nitrogen-limited state, thereby increasing the nitrogen fixation level activity. These processes may also be responsive to intracellular or extracellular levels of ammonia, urea or nitrates.

The amtB protein is also a target for genetic variation to facilitate field-based nitrogen fixation using the methods described herein. Ammonia uptake from the environment can be reduced by decreasing the expression level of amtB protein. Without intracellular ammonia, the endophyte is not able to sense the high level of ammonia, preventing the down-regulation of nitrogen fixation genes. Any ammonia that manages to get into the intracellular compartment is converted into glutamine. Intracellular glutamine level is the major currency of nitrogen sensing. Decreasing the intracellular glutamine level prevents the cells from sensing high ammonium levels in the environment. This effect can be achieved by increasing the expression level of glutaminase, an enzyme that converts glutamine into glutamate. In addition, intracellular glutamine can also be reduced by decreasing glutamine synthase (an enzyme that converts ammonia into glutamine). In diazotrophs, fixed ammonia is quickly assimilated into glutamine and glutamate to be used for cellular processes. Disruptions to ammonia assimilation may enable diversion of fixed nitrogen to be exported from the cell as ammonia. The fixed ammonia is predominantly assimilated into glutamine by glutamine synthetase (GS), encoded by glnA, and subsequently into glutamine by glutamine oxoglutarate aminotransferase (GOGAT). In some examples, glnS encodes a glutamine synthetase. GS is regulated post-translationally by GS adenylyl transferase (GlnE), a bi-functional enzyme encoded by glnE that catalyzes both the adenylylation and de-adenylylation of GS through activity of its adenylyl-transferase (AT) and adenylyl-removing (AR) domains, respectively. Under nitrogen limiting conditions, glnA is expressed, and GlnE's AR domain de-adynylylates GS, allowing it to be active. Under conditions of nitrogen excess, glnA expression is turned off, and GlnE's AT domain is activated allosterically by glutamine, causing the adenylylation and deactivation of GS.

Furthermore, the draT gene may also be a target for genetic variation to facilitate field-based nitrogen fixation using the methods described herein. Once nitrogen fixing enzymes are produced by the cell, nitrogenase shut-off represents another level in which cell downregulates fixation activity in high nitrogen condition. This shut-off could be removed by decreasing the expression level of DraT.

Methods for imparting new microbial phenotypes can be performed at the transcriptional, translational, and post-translational levels. The transcriptional level includes changes at the promoter (such as changing sigma factor affinity or binding sites for transcription factors, including deletion of all or a portion of the promoter) or changing transcription terminators and attenuators. The translational level includes changes at the ribosome binding sites and changing mRNA degradation signals. The post-translational level includes mutating an enzyme's active site and changing protein-protein interactions. These changes can be achieved in a multitude of ways. Reduction of expression level (or complete abolishment) can be achieved by swapping the native ribosome binding site (RBS) or promoter with another with lower strength/efficiency. ATG start sites can be swapped to a GTG, TTG, or CTG start codon, which results in reduction in translational activity of the coding region. Complete abolishment of expression can be done by knocking out (deleting) the coding region of a gene. Frameshifting the open reading frame (ORF) likely will result in a premature stop codon along the ORF, thereby creating a non-functional truncated product. Insertion of in-frame stop codons will also similarly create a non-functional truncated product. Addition of a degradation tag at the N or C terminal can also be done to reduce the effective concentration of a particular gene.

Conversely, expression level of the genes described herein can be achieved by using a stronger promoter. To ensure high promoter activity during high nitrogen level condition (or any other condition), a transcription profile of the whole genome in a high nitrogen level condition could be obtained and active promoters with a desired transcription level can be chosen from that dataset to replace the weak promoter. Weak start codons can be swapped out with an ATG start codon for better translation initiation efficiency. Weak ribosomal binding sites (RBS) can also be swapped out with a different RBS with higher translation initiation efficiency. In addition, site specific mutagenesis can also be performed to alter the activity of an enzyme.

Increasing the level of nitrogen fixation that occurs in a plant can lead to a reduction in the amount of chemical fertilizer needed for crop production and reduce greenhouse gas emissions (e.g., nitrous oxide).

Generation of Bacterial Populations Isolation of Bacteria

Microbes useful in methods and compositions disclosed herein can be obtained by extracting microbes from surfaces or tissues of native plants. Microbes can be obtained by grinding seeds to isolate microbes. Microbes can be obtained by planting seeds in diverse soil samples and recovering microbes from tissues. Additionally, microbes can be obtained by inoculating plants with exogenous microbes and determining which microbes appear in plant tissues. Non-limiting examples of plant tissues may include a seed, seedling, leaf, cutting, plant, bulb, or tuber.

A method of obtaining microbes may be through the isolation of bacteria from soils. Bacteria may be collected from various soil types. In some example, the soil can be characterized by traits such as high or low fertility, levels of moisture, levels of minerals, and various cropping practices. For example, the soil may be involved in a crop rotation where different crops are planted in the same soil in successive planting seasons. The sequential growth of different crops on the same soil may prevent disproportionate depletion of certain minerals. The bacteria can be isolated from the plants growing in the selected soils. The seedling plants can be harvested at 2-6 weeks of growth. For example, at least 400 isolates can be collected in a round of harvest Soil and plant types reveal the plant phenotype as well as the conditions, which allow for the downstream enrichment of certain phenotypes.

Microbes can be isolated from plant tissues to assess microbial traits. The parameters for processing tissue samples may be varied to isolate different types of associative microbes, such as rhizospheric bacteria, epiphytes, or endophytes. The isolates can be cultured in nitrogen-free media to enrich for bacteria that perform nitrogen fixation. Alternatively, microbes can be obtained from global strain banks.

In planta analytics are performed to assess microbial traits. In some embodiments, the plant tissue can be processed for screening by high throughput processing for DNA and RNA. Additionally, non-invasive measurements can be used to assess plant characteristics, such as colonization. Measurements on wild microbes can be obtained on a plant-by-plant basis. Measurements on wild microbes can also be obtained in the field using medium throughput methods. Measurements can be done successively over time. Model plant system can be used including, but not limited to, Setaria.

Microbes in a plant system can be screened via transcriptional profiling of a microbe in a plant system. Examples of screening through transcriptional profiling are using methods of quantitative polymerase chain reaction (qPCR), molecular barcodes for transcript detection, Next Generation Sequencing, and microbe tagging with fluorescent markers. Impact factors can be measured to assess colonization in the greenhouse including, but not limited to, microbiome, abiotic factors, soil conditions, oxygen, moisture, temperature, inoculum conditions, and root localization. Nitrogen fixation can be assessed in bacteria by measuring 15N gas/fertilizer (dilution) with IRMS or NanoSIMS as described herein NanoSIMS is high-resolution secondary ion mass spectrometry. The NanoSIMS technique is a way to investigate chemical activity from biological samples. The catalysis of reduction of oxidation reactions that drive the metabolism of microorganisms can be investigated at the cellular, subcellular, molecular and elemental level. NanoSIMS can provide high spatial resolution of greater than 0.1 μm. NanoSIMS can detect the use of isotope tracers such as ¹³C, ¹⁵N, and ¹⁸O. Therefore, NanoSIMS can be used to the chemical activity nitrogen in the cell.

Automated greenhouses can be used for planta analytics. Plant metrics in response to microbial exposure include, but are not limited to, biomass, chloroplast analysis, CCD camera, volumetric tomography measurements.

One way of enriching a microbe population is according to genotype. For example, a polymerase chain reaction (PCR) assay with a targeted primer or specific primer. Primers designed for the nifH gene can be used to identity diazotrophs because diazotrophs express the nifH gene in the process of nitrogen fixation. A microbial population can also be enriched via single-cell culture-independent approaches and chemotaxis-guided isolation approaches. Alternatively, targeted isolation of microbes can be performed by culturing the microbes on selection media. Premeditated approaches to enriching microbial populations for desired traits can be guided by bioinformatics data and are described herein.

Enriching for Microbes with Nitrogen Fixation Capabilities Using Bioinformatics

Bioinformatic tools can be used to identify and isolate plant growth promoting rhizobacteria (PGPRs), which are selected based on their ability to perform nitrogen fixation. Microbes with high nitrogen fixing ability can promote favorable traits in plants. Bioinformatic modes of analysis for the identification of PGPRs include, but are not limited to, genomics, metagenomics, targeted isolation, gene sequencing, transcriptome sequencing, and modeling.

Genomics analysis can be used to identify PGPRs and confirm the presence of mutations with methods of Next Generation Sequencing as described herein and microbe version control.

Metagenomics can be used to identify and isolate PGPR using a prediction algorithm for colonization. Metadata can also be used to identify the presence of an engineered strain in environmental and greenhouse samples.

Transcriptomic sequencing can be used to predict genotypes leading to PGPR phenotypes. Additionally, transcriptomic data is used to identify promoters for altering gene expression. Transcriptomic data can be analyzed in conjunction with the Whole Genome Sequence (WGS) to generate models of metabolism and gene regulatory networks.

Domestication of Microbes

Microbes isolated from nature can undergo a domestication process wherein the microbes are converted to a form that is genetically trackable and identifiable. One way to domesticate a microbe is to engineer it with antibiotic resistance. The process of engineering antibiotic resistance can begin by determining the antibiotic sensitivity in the wild type microbial strain. If the bacteria are sensitive to the antibiotic, then the antibiotic can be a good candidate for antibiotic resistance engineering. Subsequently, an antibiotic resistant gene or a counterselectable suicide vector can be incorporated into the genome of a microbe using recombineering methods. A counterselectable suicide vector may consist of a deletion of the gene of interest, a selectable marker, and the counterselectable marker sacB. Counterselection can be used to exchange native microbial DNA sequences with antibiotic resistant genes. A medium throughput method can be used to evaluate multiple microbes simultaneously allowing for parallel domestication. Alternative methods of domestication include the use of homing nucleases to prevent the suicide vector sequences from looping out or from obtaining intervening vector sequences.

DNA vectors can be introduced into bacteria via several methods including electroporation and chemical transformations. A standard library of vectors can be used for transformations. An example of a method of gene editing is CRISPR preceded by Cas9 testing to ensure activity of Cas9 in the microbes.

Non-Transgenic Engineering of Microbes

A microbial population with favorable traits can be obtained via directed evolution. Direct evolution is an approach wherein the process of natural selection is mimicked to evolve proteins or nucleic acids towards a user-defined goal. An example of direct evolution is when random mutations are introduced into a microbial population, the microbes with the most favorable traits are selected, and the growth of the selected microbes is continued. The most favorable traits in growth promoting rhizobacteria (PGPRs) may be in nitrogen fixation. The method of directed evolution may be iterative and adaptive based on the selection process after each iteration.

Plant growth promoting rhizobacteria (PGPRs) with high capability of nitrogen fixation can be generated. The evolution of PGPRs can be carried out via the introduction of genetic variation. Genetic variation can be introduced via polymerase chain reaction mutagenesis, oligonucleotide-directed mutagenesis, saturation mutagenesis, fragment shuffling mutagenesis, homologous recombination, CRISPR/Cas9 systems, chemical mutagenesis, and combinations thereof. These approaches can introduce random mutations into the microbial population. For example, mutants can be generated using synthetic DNA or RNA via oligonucleotide-directed mutagenesis. Mutants can be generated using tools contained on plasmids, which are later cured. Genes of interest can be identified using libraries from other species with improved traits including, but not limited to, improved PGPR properties, improved colonization of cereals, increased oxygen sensitivity, increased nitrogen fixation, and increased ammonia excretion. Intrageneric genes can be designed based on these libraries using software such as Geneious or Platypus design software. Mutations can be designed with the aid of machine learning. Mutations can be designed with the aid of a metabolic model. Automated design of the mutation can be done using a la Platypus and will guide RNAs for Cas-directed mutagenesis.

The intra-generic genes can be transferred into the host microbe. Additionally, reporter systems can also be transferred to the microbe. The reporter systems characterize promoters, determine the transformation success, screen mutants, and act as negative screening tools.

The microbes carrying the mutation can be cultured via serial passaging. A microbial colony contains a single variant of the microbe. Microbial colonies are screened with the aid of an automated colony picker and liquid handler. Mutants with gene duplication and increased copy number express a higher genotype of the desired trait.

Selection of Plant Growth Promoting Microbes Based on Nitrogen Fixation

The microbial colonies can be screened using various assays to assess nitrogen fixation. One way to measure nitrogen fixation is via a single fermentative assay, which measures nitrogen excretion. An alternative method is the acetylene reduction assay (ARA) with in-line sampling over time. ARA can be performed in high throughput plates of microtube arrays. ARA can be performed with live plants and plant tissues. The media formulation and media oxygen concentration can be varied in ARA assays. Another method of screening microbial variants is by using biosensors. The use of NanoSIMS and Raman microspectroscopy can be used to investigate the activity of the microbes. In some cases, bacteria can also be cultured and expanded using methods of fermentation in bioreactors. The bioreactors are designed to improve robustness of bacteria growth and to decrease the sensitivity of bacteria to oxygen. Medium to high TP plate-based microfermentors are used to evaluate oxygen sensitivity, nutritional needs, nitrogen fixation, and nitrogen excretion. The bacteria can also be co-cultured with competitive or beneficial microbes to elucidate cryptic pathways. Flow cytometry can be used to screen for bacteria that produce high levels of nitrogen using chemical, colorimetric, or fluorescent indicators. The bacteria may be cultured in the presence or absence of a nitrogen source. For example, the bacteria may be cultured with glutamine, ammonia, urea or nitrates.

Guided Microbial Remodeling—An Overview

Guided microbial remodeling is a method to systematically identify and improve the role of species within the crop microbiome. In some aspects, and according to a particular methodology of grouping/categorization, the method comprises three steps: 1) selection of candidate species by mapping plant-microbe interactions and predicting regulatory networks linked to a particular phenotype, 2) pragmatic and predictable improvement of microbial phenotypes through intra-species crossing of regulatory networks and gene clusters within a microbe's genome, and 3) screening and selection of new microbial genotypes that produce desired crop phenotypes.

To systematically assess the improvement of strains, a model is created that links colonization dynamics of the microbial community to genetic activity by key species. The model is used to predict genetic targets for non-intergeneric genetic remodeling (i.e. engineering the genetic architecture of the microbe in a non-transgenic fashion). See, FIG. 1A for a graphical representation of an embodiment of the process.

As illustrated in FIG. 1A-IO, rational improvement of the crop microbiome may be used to increase soil biodiversity, tune impact of keystone species, and/or alter timing and expression of important metabolic pathways.

To this end, the inventors have developed a platform to identify and improve the role of strains within the crop microbiome. In some aspects, the inventors call this process microbial breeding.

The aforementioned “Guided Microbial Remodeling” process will be further elaborated upon in the Examples, for instance in Example 1, entitled. “Guided Microbial Remodeling—A Platform for the Rational Improvement of Microbial Species for Agriculture.”

Serial Passage

Production of bacteria to improve plant traits (e.g., nitrogen fixation) can be achieved through serial passage. The production of this bacteria can be done by selecting plants, which have a particular improved trait that is influenced by the microbial flora, in addition to identifying bacteria and/or compositions that are capable of imparting one or more improved traits to one or more plants. One method of producing a bacteria to improve a plant trait includes the steps of: (a) isolating bacteria from tissue or soil of a first plant, (b) introducing a genetic variation into one or more of the bacteria to produce one or more variant bacteria; (c) exposing a plurality of plants to the variant bacteria; (d) isolating bacteria from tissue or soil of one of the plurality of plants, wherein the plant from which the bacteria is isolated has an improved trait relative to other plants in the plurality of plants; and (e) repeating steps (b) to (d) with bacteria isolated from the plant with an improved trait (step (d)). Steps (b) to (d) can be repeated any number of times (e.g., once, twice, three times, four times, five times, ten times, or more) until the improved trait in a plant reaches a desired level. Further, the plurality of plants can be more than two plants, such as 10 to 20 plants, or 20 or more, 50 or more, 100 or more, 300 or more, 500 or more, or 1000 or more plants.

In addition to obtaining a plant with an improved trait, a bacterial population comprising bacteria comprising one or more genetic variations introduced into one or more genes (e.g., genes regulating nitrogen fixation) is obtained. By repeating the steps described above, a population of bacteria can be obtained that include the most appropriate members of the population that correlate with a plant trait of interest. The bacteria in this population can be identified and their beneficial properties determined, such as by genetic and/or phenotypic analysis. Genetic analysis may occur of isolated bacteria in step (a). Phenotypic and/or genotypic information may be obtained using techniques including, high through-put screening of chemical components of plant origin, sequencing techniques including high throughput sequencing of genetic material, differential display techniques (including DDRT-PCR, and DD-PCR), nucleic acid microarray techniques, RNA-sequencing (Whole Transcriptome Shotgun Sequencing), and qRT-PCR (quantitative real time PCR). Information gained can be used to obtain community profiling information on the identity and activity of bacteria present, such as phylogenetic analysis or microarray-based screening of nucleic acids coding for components of rRNA operons or other taxonomically informative loci. Examples of taxonomically informative loci include 16S rRNA gene, 23S rRNA gene, 5S rRNA gene, 5.8S rRNA gene, 12S rRNA gene, 18S rRNA gene, 28S rRNA gene, gyrB gene, rpoB gene, fusA gene, recA gene, coxl gene, nifD gene. Example processes of taxonomic profiling to determine taxa present in a population are described in US20140155283. Bacterial identification may comprise characterizing activity of one or more genes or one or more signaling pathways, such as genes associated with the nitrogen fixation pathway. Synergistic interactions (where two components, by virtue of their combination, increase a desired effect by more than an additive amount) between different bacterial species may also be present in the bacterial populations.

Genetic Variation—Locations and Sources of Genomic Alteration

The genetic variation may be a gene selected from the group consisting of: nifA, nifL, ntrB, ntrC, glnA, glnB, glnK, draT, amtB, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, niff, nifB, and nifQ. The genetic variation may be a variation in a gene encoding a protein with functionality selected from the group consisting of: glutamine synthetase, glutaminase, glutamine synthetase adenylyltransferase, transcriptional activator, anti-transcriptional activator, pyruvate flavodoxin oxidoreductase, flavodoxin, or NAD+-dinitrogen-reductase aDP-D-ribosyltransferase. The genetic variation may be a mutation that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; or decreased uridylyl-removing activity of GlnD. Introducing a genetic variation may comprise insertion and/or deletion of one or more nucleotides at a target site, such as 1, 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or more nucleotides. The genetic variation introduced into one or more bacteria of the methods disclosed herein may be a knock-out mutation (e.g. deletion of a promoter, insertion or deletion to produce a premature stop codon, deletion of an entire gene), or it may be elimination or abolishment of activity of a protein domain (e.g. point mutation affecting an active site, or deletion of a portion of a gene encoding the relevant portion of the protein product), or it may alter or abolish a regulatory sequence of a target gene. One or more regulatory sequences may also be inserted, including heterologous regulatory sequences and regulatory sequences found within a genome of a bacterial species or genus corresponding to the bacteria into which the genetic variation is introduced. Moreover, regulatory sequences may be selected based on the expression level of a gene in a bacterial culture or within a plant tissue. The genetic variation may be a pre-determined genetic variation that is specifically introduced to a target site. The genetic variation may be a random mutation within the target site. The genetic variation may be an insertion or deletion of one or more nucleotides. In some cases, a plurality of different genetic variations (e.g. 2, 3, 4, 5, 10, or more) are introduced into one or more of the isolated bacteria before exposing the bacteria to plants for assessing trait improvement. The plurality of genetic variations can be any of the above types, the same or different types, and in any combination. In some cases, a plurality of different genetic variations are introduced serially, introducing a first genetic variation after a first isolation step, a second genetic variation after a second isolation step, and so forth so as to accumulate a plurality of genetic variations in bacteria imparting progressively improved traits on the associated plants.

Genetic Variation—Methods of Introducing Genomic Alteration

In general, the term “genetic variation” refers to any change introduced into a polynucleotide sequence relative to a reference polynucleotide, such as a reference genome or portion thereof, or reference gene or portion thereof. A genetic variation may be referred to as a “mutation,” and a sequence or organism comprising a genetic variation may be referred to as a “genetic variant” or “mutant”. Genetic variations can have any number of effects, such as the increase or decrease of some biological activity, including gene expression, metabolism, and cell signaling. Genetic variations can be specifically introduced to a target site, or introduced randomly. A variety of molecular tools and methods are available for introducing genetic variation. For example, genetic variation can be introduced via polymerase chain reaction mutagenesis, oligonucleotide-directed mutagenesis, saturation mutagenesis, fragment shuffling mutagenesis, homologous recombination, recombineering, lambda red mediated recombination, CRISPR/Cas9 systems, chemical mutagenesis, and combinations thereof. Chemical methods of introducing genetic variation include exposure of DNA to a chemical mutagen, e.g., ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-nitrosourea (EN U), N-methyl-N-nitro-N′-nitrosoguanidine, 4-nitroquinoline N-oxide, diethylsulfate, benzopyrene, cyclophosphamide, bleomycin, triethylmelamine, acrylamide monomer, nitrogen mustard, vincristine, diepoxyalkanes (for example, diepoxy butane), ICR-170, formaldehyde, procarbazine hydrochloride, ethylene oxide, dimethylnitrosamine, 7,12 dimethylbenz(a)anthracene, chlorambucil, hexamethylphosphoramide, bisulfan, and the like. Radiation mutation-inducing agents include ultraviolet radiation, γ-irradiation, X-rays, and fast neutron bombardment. Genetic variation can also be introduced into a nucleic acid using, e.g., trimethylpsoralen with ultraviolet light. Random or targeted insertion of a mobile DNA element, e.g., a transposable element, is another suitable method for generating genetic variation. Genetic variations can be introduced into a nucleic acid during amplification in a cell-free in vitro system, e.g., using a polymerase chain reaction (PCR) technique such as error-prone PCR. Genetic variations can be introduced into a nucleic acid in vitro using DNA shuffling techniques (e.g., exon shuffling, domain swapping, and the like). Genetic variations can also be introduced into a nucleic acid as a result of a deficiency in a DNA repair enzyme in a cell, e.g., the presence in a cell of a mutant gene encoding a mutant DNA repair enzyme is expected to generate a high frequency of mutations (i.e., about 1 mutation/100 genes-1 mutation/10,000 genes) in the genome of the cell. Examples of genes encoding DNA repair enzymes include but are not limited to Mut H, Mut S, Mut L, and Mut U, and the homologs thereof in other species (e.g., MSH 1 6, PMS 1 2, MLH 1, GTBP, ERCC-1, and the like). Example descriptions of various methods for introducing genetic variations are provided in e.g., Stemple (2004) Nature 5:1-7; Chiang et al. (1993) PCR Methods Appl 2(3): 210-217; Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; and U.S. Pat. Nos. 6,033,861, and 6,773,900.

Genetic variations introduced into microbes may be classified as transgenic, cisgenic, intragenomic, intrageneric, intergeneric, synthetic, evolved, rearranged, or SNPs.

Genetic variation may be introduced into numerous metabolic pathways within microbes to elicit improvements in the traits described above. Representative pathways include sulfur uptake pathways, glycogen biosynthesis, the glutamine regulation pathway, the molybdenum uptake pathway, the nitrogen fixation pathway, ammonia assimilation, ammonia excretion or secretion, nitrogen uptake, glutamine biosynthesis, annamox, phosphate solubilization, organic acid transport, organic acid production, agglutinins production, reactive oxygen radical scavenging genes, Indole Acetic Acid biosynthesis, trehalose biosynthesis, plant cell wall degrading enzymes or pathways, root attachment genes, exopolysaccharide secretion, glutamate synthase pathway, iron uptake pathways, siderophore pathway, chitinase pathway, ACC deaminase, glutathione biosynthesis, phosphorous signaling genes, quorum quenching pathway, cytochrome pathways, hemoglobin pathway, bacterial hemoglobin-like pathway, small RNA rsmZ, rhizobitoxine biosynthesis, lapA adhesion protein, AHL quorum sensing pathway, phenazine biosynthesis, cyclic lipopeptide biosynthesis, and antibiotic production.

CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats)/CRISPR-associated (Cas) systems can be used to introduce desired mutations. CRISPR/Cas9 provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. The Cas9 protein (or functional equivalent and/or variant thereof, i.e., Cas9-like protein) naturally contains DNA endonuclease activity that depends on the association of the protein with two naturally occurring or synthetic RNA molecules called crRNA and tracrRNA (also called guide RNAs). In some cases, the two molecules are covalently link to form a single molecule (also called a single guide RNA (“sgRNA”). Thus, the Cas9 or Cas9-like protein associates with a DNA-targeting RNA (which term encompasses both the two-molecule guide RNA configuration and the single-molecule guide RNA configuration), which activates the Cas9 or Cas9-like protein and guides the protein to a target nucleic acid sequence. If the Cas9 or Cas9-like protein retains its natural enzymatic function, it will cleave target DNA to create a double-stranded break, which can lead to genome alteration (i.e., editing: deletion, insertion (when a donor polynucleotide is present), replacement, etc.), thereby altering gene expression. Some variants of Cas9 (which variants are encompassed by the term Cas9-like) have been altered such that they have a decreased DNA cleaving activity (in some cases, they cleave a single strand instead of both strands of the target DNA, while in other cases, they have severely reduced to no DNA cleavage activity). Further exemplary descriptions of CRISPR systems for introducing genetic variation can be found in, e.g. U.S. Pat. No. 8,795,965.

As a cyclic amplification technique, polymerase chain reaction (PCR) mutagenesis uses mutagenic primers to introduce desired mutations. PCR is performed by cycles of denaturation, annealing, and extension. After amplification by PCR, selection of mutated DNA and removal of parental plasmid DNA can be accomplished by: 1) replacement of dCTP by hydroxymethylated-dCTP during PCR, followed by digestion with restriction enzymes to remove non-hydroxymethylated parent DNA only; 2) simultaneous mutagenesis of both an antibiotic resistance gene and the studied gene changing the plasmid to a different antibiotic resistance, the new antibiotic resistance facilitating the selection of the desired mutation thereafter; 3) after introducing a desired mutation, digestion of the parent methylated template DNA by restriction enzyme Dpn1 which cleaves only methylated DNA, by which the mutagenized unmethylated chains are recovered; or 4) circularization of the mutated PCR products in an additional ligation reaction to increase the transformation efficiency of mutated DNA. Further description of exemplary methods can be found in e.g. U.S. Pat. Nos. 7,132,265, 6,713,285, 6,673,610, 6,391,548, 5,789,166, 5,780,270, 5,354,670, 5,071,743, and US20100267147.

Oligonucleotide-directed mutagenesis, also called site-directed mutagenesis, typically utilizes a synthetic DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so that it can hybridize with the DNA in the gene of interest. The mutation may be a single base change (a point mutation), multiple base changes, deletion, or insertion, or a combination of these. The single-strand primer is then extended using a DNA polymerase, which copies the rest of the gene. The gene thus copied contains the mutated site, and may then be introduced into a host cell as a vector and cloned. Finally, mutants can be selected by DNA sequencing to check that they contain the desired mutation.

Genetic variations can be introduced using error-prone PCR. In this technique the gene of interest is amplified using a DNA polymerase under conditions that are deficient in the fidelity of replication of sequence. The result is that the amplification products contain at least one error in the sequence. When a gene is amplified and the resulting produces) of the reaction contain one or more alterations in sequence when compared to the template molecule, the resulting products are mutagenized as compared to the template. Another means of introducing random mutations is exposing cells to a chemical mutagen, such as nitrosoguanidine or ethyl methanesulfonate (Nestmann, Mutat Res 1975 June; 28(3):323-30), and the vector containing the gene is then isolated from the host.

Saturation mutagenesis is another form of random mutagenesis, in which one tries to generate all or nearly all possible mutations at a specific site, or narrow region of a gene. In a general sense, saturation mutagenesis is comprised of mutagenizing a complete set of mutagenic cassettes (wherein each cassette is, for example, 1-500 bases in length) in defined polynucleotide sequence to be mutagenized (wherein the sequence to be mutagenized is, for example, from 15 to 100,000 bases in length). Therefore, a group of mutations (e.g. ranging from 1 to 100 mutations) is introduced into each cassette to be mutagenized. A grouping of mutations to be introduced into one cassette can be different or the same from a second grouping of mutations to be introduced into a second cassette during the application of one round of saturation mutagenesis. Such groupings are exemplified by deletions, additions, groupings of particular codons, and groupings of particular nucleotide cassettes.

Fragment shuffling mutagenesis, also called DNA shuffling, is a way to rapidly propagate beneficial mutations. In an example of a shuffling process, DNAse is used to fragment a set of parent genes into pieces of e.g. about 50-100 bp in length. This is then followed by a polymerase chain reaction (PCR) without primers—DNA fragments with sufficient overlapping homologous sequence will anneal to each other and are then be extended by DNA polymerase. Several rounds of this PCR extension are allowed to occur, after some of the DNA molecules reach the size of the parental genes. These genes can then be amplified with another PCR, this time with the addition of primers that are designed to complement the ends of the strands. The primers may have additional sequences added to their 5′ ends, such as sequences for restriction enzyme recognition sites needed for ligation into a cloning vector. Further examples of shuffling techniques are provided in US20050266541.

Homologous recombination mutagenesis involves recombination between an exogenous DNA fragment and the targeted polynucleotide sequence. After a double-stranded break occurs, sections of DNA around the 5′ ends of the break are cut away in a process called resection. In the strand invasion step that follows, an overhanging 3′ end of the broken DNA molecule then “invades” a similar or identical DNA molecule that is not broken. The method can be used to delete a gene, remove exons, add a gene, and introduce point mutations. Homologous recombination mutagenesis can be permanent or conditional. Typically, a recombination template is also provided. A recombination template may be a component of another vector, contained in a separate vector, or provided as a separate polynucleotide. In some embodiments, a recombination template is designed to serve as a template in homologous recombination, such as within or near a target sequence nicked or cleaved by a site-specific nuclease. A template polynucleotide may be of any suitable length, such as about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length. In some embodiments, the template polynucleotide is complementary to a portion of a polynucleotide comprising the target sequence. When optimally aligned, a template polynucleotide might overlap with one or more nucleotides of a target sequences (e.g. about or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides). In some embodiments, when a template sequence and a polynucleotide comprising a target sequence are optimally aligned, the nearest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence. Non-limiting examples of site-directed nucleases useful in methods of homologous recombination include zinc finger nucleases, CRISPR nucleases, TALE nucleases, and meganuclease. For a further description of the use of such nucleases, see e.g. U.S. Pat. No. 8,795,965 and US20140301990.

Mutagens that create primarily point mutations and short deletions, insertions, transversions, and/or transitions, including chemical mutagens or radiation, may be used to create genetic variations. Mutagens include, but are not limited to, ethyl methanesulfonate, methylmethane sulfonate, N-ethyl-N-nitrosurea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N′-nitro-Nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12 dimethyl-benz(a)anthracene, ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane, diepoxybutane, and the like), 2-methoxy-6-chloro-9[3-(ethyl-2-chloro-ethyl)aminopropylamino]acridine dihydrochloride and formaldehyde.

Introducing genetic variation may be an incomplete process, such that some bacteria in a treated population of bacteria carry a desired mutation while others do not. In some cases, it is desirable to apply a selection pressure so as to enrich for bacteria carrying a desired genetic variation. Traditionally, selection for successful genetic variants involved selection for or against some functionality imparted or abolished by the genetic variation, such as in the case of inserting antibiotic resistance gene or abolishing a metabolic activity capable of converting a non-lethal compound into a lethal metabolite. It is also possible to apply a selection pressure based on a polynucleotide sequence itself, such that only a desired genetic variation need be introduced (e.g. without also requiring a selectable marker). In this case, the selection pressure can comprise cleaving genomes lacking the genetic variation introduced to a target site, such that selection is effectively directed against the reference sequence into which the genetic variation is sought to be introduced. Typically, cleavage occurs within 100 nucleotides of the target site (e.g. within 75, 50, 25, 10, or fewer nucleotides from the target site, including cleavage at or within the target site). Cleaving may be directed by a site-specific nuclease selected from the group consisting of a Zinc Finger nuclease, a CRISPR nuclease, a TALE nuclease (TALEN), or a meganuclease. Such a process is similar to processes for enhancing homologous recombination at a target site, except that no template for homologous recombination is provided. As a result, bacteria lacking the desired genetic variation are more likely to undergo cleavage that, left unrepaired, results in cell death. Bacteria surviving selection may then be isolated for use in exposing to plants for assessing conferral of an improved trait.

A CRISPR nuclease may be used as the site-specific nuclease to direct cleavage to a target site. An improved selection of mutated microbes can be obtained by using Cas9 to kill non-mutated cells. Plants are then inoculated with the mutated microbes to re-confirm symbiosis and create evolutionary pressure to select for efficient symbionts. Microbes can then be re-isolated from plant tissues. CRISPR nuclease systems employed for selection against non-variants can employ similar elements to those described above with respect to introducing genetic variation, except that no template for homologous recombination is provided. Cleavage directed to the target site thus enhances death of affected cells.

Other options for specifically inducing cleavage at a target site are available, such as zinc finger nucleases, TALE nuclease (TALEN) systems, and meganuclease. Zinc-finger nucleases (ZFNs) are artificial DNA endonucleases generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. ZFNs can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to cleave unique target sequences. When introduced into a cell, ZFNs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double stranded breaks. Transcription activator-like effector nucleases (TALENs) are artificial DNA endonucleases generated by fusing a TAL (Transcription activator-like) effector DNA binding domain to a DNA cleavage domain. TALENS can be quickly engineered to bind practically any desired DNA sequence and when introduced into a cell, TALENs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks. Meganucleases (homing endonuclease) are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs. Meganucleases can be used to replace, eliminate or modify sequences in a highly targeted way. By modifying their recognition sequence through protein engineering, the targeted sequence can be changed. Meganucleases can be used to modify all genome types, whether bacterial, plant or animal and are commonly grouped into four families: the LAGLIDADG family (SEQ ID NO: 1), the GIY-YIG family, the His-Cyst box family and the HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII

Genetic Variation—Methods of Identification

The microbes of the present disclosure may be identified by one or more genetic modifications or alterations, which have been introduced into said microbe. One method by which said genetic modification or alteration can be identified is via reference to a SEQ ID NO that contains a portion of the microbe's genomic sequence that is sufficient to identify the genetic modification or alteration.

Further, in the case of microbes that have not had a genetic modification or alteration (e.g. a wild type, WT) introduced into their genomes, the disclosure can utilize 16S nucleic acid sequences to identify said microbes. A 16S nucleic acid sequence is an example of a “molecular marker” or “genetic marker,” which refers to an indicator that is used in methods for visualizing differences in characteristics of nucleic acid sequences. Examples of other such indicators are restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), insertion mutations, microsatellite markers (SSRs), sequence-characterized amplified regions (SCARs), cleaved amplified polymorphic sequence (CAPS) markers or isozyme markers or combinations of the markers described herein which defines a specific genetic and chromosomal location. Markers further include polynucleotide sequences encoding 16S or 18S rRNA, and internal transcribed spacer (ITS) sequences, which are sequences found between small-subunit and large-subunit rRNA genes that have proven to be especially useful in elucidating relationships or distinctions when compared against one another. Furthermore, the disclosure utilizes unique sequences found in genes of interest (e.g. nif H,D,K,L,A, glnE, amtB, etc.) to identify microbes disclosed herein.

The primary structure of major rRNA subunit 16S comprise a particular combination of conserved, variable, and hypervariable regions that evolve at different rates and enable the resolution of both very ancient lineages such as domains, and more modern lineages such as genera. The secondary structure of the 16S subunit include approximately 50 helices which result in base pairing of about 67% of the residues. These highly conserved secondary structural features are of great functional importance and can be used to ensure positional homology in multiple sequence alignments and phylogenetic analysis. Over the previous few decades, the 16S rRNA gene has become the most sequenced taxonomic marker and is the cornerstone for the current systematic classification of bacteria and archaea (Yarza et al. 2014. Nature Rev. Micro. 12:635-45).

Thus, in certain aspects, the disclosure provides for a sequence, which shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence in Tables 23, 24, 25, and 26.

Thus, in certain aspects, the disclosure provides for a microbe that comprises a sequence, which shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 62-303. These sequences and their associated descriptions can be found in Tables 25 and 26.

In some aspects, the disclosure provides for a microbe that comprises a 16S nucleic acid sequence, which shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs. 85, 96, 111, 121, 122, 123, 124, 136, 149, 157, 167, 261, 262, 269, 277-283. These sequences and their associated descriptions can be found in Table 26.

In some aspects, the disclosure provides for a microbe that comprises a nucleic acid sequence, which shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs. 86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276, 284-295. These sequences and their associated descriptions can be found in Table 26.

In some aspects, the disclosure provides for a microbe that comprises a nucleic acid sequence, which shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 177-260, 296-303. These sequences and their associated descriptions can be found in Table 26.

In some aspects, the disclosure provides for a microbe that comprises, or primer that comprises, or probe that comprises, or non-native junction sequence that comprises, a nucleic acid sequence, which shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs. 304-424. These sequences are described in Table 27.

In some aspects, the disclosure provides for a microbe that comprises a non-native junction sequence that shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs. 372-405. These sequences are described in Table 27.

In some aspects, the disclosure provides for a microbe that comprises an amino acid sequence, which shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 77, 78, 81, 82, or 83. These sequences and their associated descriptions can be found in Table 25.

Genetic Variation—Methods of Detection: Primers, Probes, and Assays

The present disclosure teaches primers, probes, and assays that are useful for detecting the microbes taught herein. In some aspects, the disclosure provides for methods of detecting the WT parental strains. In other aspects, the disclosure provides for methods of detecting the non-intergeneric engineered microbes derived from the WT strains. In aspects, the present disclosure provides methods of identifying non-intergeneric genetic alterations in a microbe.

In aspects, the genomic engineering methods of the present disclosure lead to the creation of non-natural nucleotide “junction” sequences in the derived non-intergeneric microbes. These non-naturally occurring nucleotide junctions can be used as a type of diagnostic that is indicative of the presence of a particular genetic alteration in a microbe taught herein.

The present techniques are able to detect these non-naturally occurring nucleotide junctions via the utilization of specialized quantitative PCR methods, including uniquely designed primers and probes. In some aspects, the probes of the disclosure bind to the non-naturally occurring nucleotide junction sequences. In some aspects, traditional PCR is utilized. In other aspects, real-time PCR is utilized. In some aspects, quantitative PCR (qPCR) is utilized.

Thus, the disclosure can cover the utilization of two common methods for the detection of PCR products in real-time: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence. In some aspects, only the non-naturally occurring nucleotide junction will be amplified via the taught primers, and consequently can be detected via either a non-specific dye, or via the utilization of a specific hybridization probe. In other aspects, the primers of the disclosure are chosen such that the primers flank either side of a junction sequence, such that if an amplification reaction occurs, then said junction sequence is present.

Aspects of the disclosure involve non-naturally occurring nucleotide junction sequence molecules per se, along with other nucleotide molecules that are capable of binding to said non-naturally occurring nucleotide junction sequences under mild to stringent hybridization conditions. In some aspects, the nucleotide molecules that are capable of binding to said non-naturally occurring nucleotide junction sequences under mild to stringent hybridization conditions are termed “nucleotide probes.”

In aspects, genomic DNA can be extracted from samples and used to quantify the presence of microbes of the disclosure by using qPCR. The primers utilized in the qPCR reaction can be primers designed by Primer Blast (www.ncbi.nlm.nih.gov/tools/primer-blast/) to amplify unique regions of the wild-type genome or unique regions of the engineered non-intergeneric mutant strains. The qPCR reaction can be carried out using the SYBR GreenER qPCR SuperMix Universal (Thermo Fisher P/N 11762100) kit, using only forward and reverse amplification primers, alternatively, the Kapa Probe Force kit (Kapa Biosystems P/N KK4301) can be used with amplification primers and a TaqMan probe containing a FAM dye label at the 5′ end, an internal ZEN quencher, and a minor groove binder and fluorescent quencher at the 3′ end (Integrated DNA Technologies).

Certain primer, probe, and non-native junction sequences are listed in Table 27. qPCR reaction efficiency can be measured using a standard curve generated from a known quantity of gDNA from the target genome. Data can be normalized to genome copies per g fresh weight using the tissue weight and extraction volume.

Quantitative polymerase chain reaction (qPCR) is a method of quantifying, in real time, the amplification of one or more nucleic acid sequences. The real time quantification of the PCR assay permits determination of the quantity of nucleic acids being generated by the PCR amplification steps by comparing the amplifying nucleic acids of interest and an appropriate control nucleic acid sequence, which may act as a calibration standard.

TaqMan probes are often utilized in qPCR assays that require an increased specificity for quantifying target nucleic acid sequences. TaqMan probes comprise an oligonucleotide probe with a fluorophore attached to the 5′ end and a quencher attached to the 3′ end of the probe. When the TaqMan probes remain as is with the 5′ and 3′ ends of the probe in close contact with each other, the quencher prevents fluorescent signal transmission from the fluorophore. TaqMan probes are designed to anneal within a nucleic acid region amplified by a specific set of primers. As the Taq polymerase extends the primer and synthesizes the nascent strand, the 5′ to 3′ exonuclease activity of the Taq polymerase degrades the probe that annealed to the template. This probe degradation releases the fluorophore, thus breaking the close proximity to the quencher and allowing fluorescence of the fluorophore. Fluorescence detected in the qPCR assay is directly proportional to the fluorophore released and the amount of DNA template present in the reaction.

The features of qPCR allow the practitioner to eliminate the labor-intensive post-amplification step of gel electrophoresis preparation, which is generally required for observation of the amplified products of traditional PCR assays. The benefits of qPCR over conventional PCR are considerable, and include increased speed, ease of use, reproducibility, and quantitative ability.

Improvement of Traits

Methods of the present disclosure may be employed to introduce or improve one or more of a variety of desirable traits. Examples of traits that may introduced or improved include, root biomass, root length, height, shoot length, leaf number, water use efficiency, overall biomass, yield, fruit size, grain size, photosynthesis rate, tolerance to drought, heat tolerance, salt tolerance, resistance to nematode stress, resistance to a fungal pathogen, resistance to a bacterial pathogen, resistance to a viral pathogen, level of a metabolite, and proteome expression. The desirable traits, including height, overall biomass, root and/or shoot biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, seed/fruit number or mass, plant grain or fruit yield, leaf chlorophyll content, photosynthetic rate, root length, or any combination thereof, can be used to measure growth, and compared with the growth rate of reference agricultural plants (e.g., plants without the improved traits) grown under identical conditions.

A preferred trait to be introduced or improved is nitrogen fixation, as described herein. In some cases, a plant resulting from the methods described herein exhibits a difference in the trait that is at least about 5% greater, for example at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 80%, at least about 90%, or at least 100%, at least about 200%, at least about 300%, at least about 400% or greater than a reference agricultural plant grown under the same conditions in the soil. In additional examples, a plant resulting from the methods described herein exhibits a difference in the trait that is at least about 5% greater, for example at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 80%, at least about 90%, or at least 100%, at least about 200%, at least about 300%, at least about 400% or greater than a reference agricultural plant grown under similar conditions in the soil.

The trait to be improved may be assessed under conditions including the application of one or more biotic or abiotic stressors. Examples of stressors include abiotic stresses (such as heat stress, salt stress, drought stress, cold stress, and low nutrient stress) and biotic stresses (such as nematode stress, insect herbivory stress, fungal pathogen stress, bacterial pathogen stress, and viral pathogen stress).

The trait improved by methods and compositions of the present disclosure may be nitrogen fixation, including in a plant not previously capable of nitrogen fixation. In some cases, bacteria isolated according to a method described herein produce 1% or more (e.g. 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or more) of a plant's nitrogen, which may represent an increase in nitrogen fixation capability of at least 2-fold (e.g. 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold, or more) as compared to bacteria isolated from the first plant before introducing any genetic variation. In some cases, the bacteria produce 5% or more of a plant's nitrogen. The desired level of nitrogen fixation may be achieved after repeating the steps of introducing genetic variation, exposure to a plurality of plants, and isolating bacteria from plants with an improved trait one or more times (e.g. 1, 2, 3, 4, 5, 10, 15, 25, or more times). In some cases, enhanced levels of nitrogen fixation are achieved in the presence of fertilizer supplemented with glutamine, ammonia, or other chemical source of nitrogen. Methods for assessing degree of nitrogen fixation are known, examples of which are described herein.

Microbe breeding is a method to systematically identify and improve the role of species within the crop microbiome. The method comprises three steps: 1) selection of candidate species by mapping plant-microbe interactions and predicting regulatory networks linked to a particular phenotype, 2) pragmatic and predictable improvement of microbial phenotypes through intra-species crossing of regulatory networks and gene clusters, and 3) screening and selection of new microbial genotypes that produce desired crop phenotypes. To systematically assess the improvement of strains, a model is created that links colonization dynamics of the microbial community to genetic activity by key species. The model is used to predict genetic targets for breeding and improve the frequency of selecting improvements in microbiome-encoded traits of agronomic relevance.

Measuring Nitrogen Delivered in an Agriculturally Relevant Field Context

In the field, the amount of nitrogen delivered can be determined by the function of colonization multiplied by the activity.

${{Nitrogen}\mspace{14mu}{delivered}} = {\int\limits_{{{Time}\mspace{14mu}\&}\mspace{14mu}{Space}}{{Colonization} \times {Activity}}}$

The above equation requires (1) the average colonization per unit of plant tissue, and (2) the activity as either the amount of nitrogen fixed or the amount of ammonia excreted by each microbial cell. To convert to pounds of nitrogen per acre, corn growth physiology is tracked over time, e.g., size of the plant and associated root system throughout the maturity stages.

The pounds of nitrogen delivered to a crop per acre-season can be calculated by the following equation:

${{Nitrogen}\mspace{14mu}{delivered}} = {\int{{Plant}\mspace{14mu}{{Tissue}(t)} \times {{Colonization}(t)} \times {{Activity}(t)}\underset{\_}{dt}}}$

The Plant Tissue (t) is the fresh weight of corn plant tissue over the growing time (t). Values for reasonably making the calculation are described in detail in the publication entitled Roots, Growth and Nutrient Uptake (Mengel. Dept, of Agronomy Pub. #AGRY-95-08 (Rev. May-95, p. 1-8).

The Colonization (t) is the amount of the microbes of interest found within the plant tissue, per gram fresh weight of plant tissue, at any particular time, t, during the growing season. In the instance of only a single time point available, the single time point is normalized as the peak colonization rate over the season, and the colonization rate of the remaining time points are adjusted accordingly.

Activity (t) is the rate of which N is fixed by the microbes of interest per unit time, at any particular time, t, during the growing season. In the embodiments disclosed herein, this activity rate is approximated by in vitro acetylene reduction assay (ARA) in ARA media in the presence of 5 mM glutamine or Ammonium excretion assay in ARA media in the presence of 5 mM ammonium ions.

The Nitrogen delivered amount is then calculated by numerically integrating the above function. In cases where the values of the variables described above are discretely measured at set time points, the values in between those time points are approximated by performing linear interpolation.

Nitrogen Fixation

Described herein are methods of increasing nitrogen fixation in a plant, comprising exposing the plant to bacteria comprising one or more genetic variations introduced into one or more genes regulating nitrogen fixation, wherein the bacteria produce 1% or more of nitrogen in the plant (e.g. 2%, 5%, 10%, or more), which may represent a nitrogen-fixation capability of at least 2-fold as compared to the plant in the absence of the bacteria. The bacteria may produce the nitrogen in the presence of fertilizer supplemented with glutamine, urea, nitrates or ammonia. Genetic variations can be any genetic variation described herein, including examples provided above, in any number and any combination. The genetic variation may be introduced into a gene selected from the group consisting of nifA, nifL, ntrB, ntrC, glutamine synthetase, glnA, glnB, glnK, draT, amtB, glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, and nifQ. The genetic variation may be a mutation that results in one or more of: increased expression or activity of nifA or glutaminase; decreased expression or activity of nifL, ntrB, glutamine synthetase, glnB, glnK, draT, amtB; decreased adenylyl-removing activity of GlnE; or decreased uridylyl-removing activity of GlnD. The genetic variation introduced into one or more bacteria of the methods disclosed herein may be a knock-out mutation or it may abolish a regulatory sequence of a target gene, or it may comprise insertion of a heterologous regulatory sequence, for example, insertion of a regulatory sequence found within the genome of the same bacterial species or genus. The regulatory sequence can be chosen based on the expression level of a gene in a bacterial culture or within plant tissue. The genetic variation may be produced by chemical mutagenesis. The plants grown in step (c) may be exposed to biotic or abiotic stressors.

The amount of nitrogen fixation that occurs in the plants described herein may be measured in several ways, for example by an acetylene-reduction (AR) assay. An acetylene-reduction assay can be performed in vitro or in vivo. Evidence that a particular bacterium is providing fixed nitrogen to a plant can include: 1) total plant N significantly increases upon inoculation, preferably with a concomitant increase in N concentration in the plant; 2) nitrogen deficiency symptoms are relieved under N-limiting conditions upon inoculation (which should include an increase in dry matter), 3) N₂ fixation is documented through the use of an ¹⁵N approach (which can be isotope dilution experiments, ¹⁵N₂ reduction assays, or ¹⁵N natural abundance assays); 4) fixed N is incorporated into a plant protein or metabolite; and 5) all of these effects are not be seen in non-inoculated plants or in plants inoculated with a mutant of the inoculum strain.

The wild-type nitrogen fixation regulatory cascade can be represented as a digital logic circuit where the inputs O₂ and NH₄ ⁺ pass through a NOR gate, the output of which enters an AND gate in addition to ATP. In some embodiments, the methods disclosed herein disrupt the influence of NH₄ ⁺ on this circuit, at multiple points in the regulatory cascade, so that microbes can produce nitrogen even in fertilized fields. However, the methods disclosed herein also envision altering the impact of ATP or O₂ on the circuitry, or replacing the circuitry with other regulatory cascades in the cell, or altering genetic circuits other than nitrogen fixation. Gene clusters can be re-engineered to generate functional products under the control of a heterologous regulatory system. By eliminating native regulatory elements outside of, and within, coding sequences of gene clusters, and replacing them with alternative regulatory systems, the functional products of complex genetic operons and other gene clusters can be controlled and/or moved to heterologous cells, including cells of different species other than the species from which the native genes were derived. Once re-engineered, the synthetic gene clusters can be controlled by genetic circuits or other inducible regulatory systems, thereby controlling the products' expression as desired. The expression cassettes can be designed to act as logic gates, pulse generators, oscillators, switches, or memory devices. The controlling expression cassette can be linked to a promoter such that the expression cassette functions as an environmental sensor, such as an oxygen, temperature, touch, osmotic stress, membrane stress, or redox sensor.

As an example, the nifL, nifA, nifT, and nifX genes can be eliminated from the nif gene cluster. Synthetic genes can be designed by codon randomizing the DNA encoding each amino acid sequence. Codon selection is performed, specifying that codon usage be as divergent as possible from the codon usage in the native gene. Proposed sequences are scanned for any undesired features, such as restriction enzyme recognition sites, transposon recognition sites, repetitive sequences, sigma 54 and sigma 70 promoters, cryptic ribosome binding sites, and rho independent terminators. Synthetic ribosome binding sites are chosen to match the strength of each corresponding native ribosome binding site, such as by constructing a fluorescent reporter plasmid in which the 150 bp surrounding a gene's start codon (from −60 to +90) is fused to a fluorescent gene. This chimera can be expressed under control of the Ptac promoter, and fluorescence measured via flow cytometry. To generate synthetic ribosome binding sites, a library of reporter plasmids using 150 bp (−60 to +90) of a synthetic expression cassette is generated. Briefly, a synthetic expression cassette can consist of a random DNA spacer, a degenerate sequence encoding an RBS library, and the coding sequence for each synthetic gene. Multiple clones are screened to identify the synthetic ribosome binding site that best matched the native ribosome binding site. Synthetic operons that consist of the same genes as the native operons are thus constructed and tested for functional complementation. A further exemplary description of synthetic operons is provided in US20140329326.

Bacterial Species

Microbes useful in the methods and compositions disclosed herein may be obtained from any source. In some cases, microbes may be bacteria, archaea, protozoa or fungi. The microbes of this disclosure may be nitrogen fixing microbes, for example a nitrogen fixing bacteria, nitrogen fixing archaea, nitrogen fixing fungi, nitrogen fixing yeast, or nitrogen fixing protozoa. Microbes useful in the methods and compositions disclosed herein may be spore forming microbes, for example spore forming bacteria. In some cases, bacteria useful in the methods and compositions disclosed herein may be Gram positive bacteria or Gram negative bacteria. In some cases, the bacteria may be an endospore forming bacteria of the Firmicute phylum. In some cases, the bacteria may be a diazotroph. In some cases, the bacteria may not be a diazotroph.

The methods and compositions of this disclosure may be used with an archaea, such as, for example, Methanothermobacter thermoautotrophicus.

In some cases, bacteria which may be useful include, but are not limited to, Agrobacterium radiobacter, Bacillus acidocaldarius, Bacillus acidoterrestris, Bacillus agri, Bacillus aizawai, Bacillus albolactis, Bacillus alcalophilus, Bacillus alvei, Bacillus aminoglucosidicus, Bacillus aminovorans, Bacillus amylolyticus (also known as Paenibacillus amylolyticus) Bacillus amyloliquefaciens, Bacillus aneurinolyticus, Bacillus atrophaeus, Bacillus azotoformans, Bacillus badius, Bacillus cereus (synonyms: Bacillus endorhythmos, Bacillus medusa), Bacillus chitinosporus, Bacillus circulans, Bacillus coagulans, Bacillus endoparasiticus Bacillus fastidiosus, Bacillus firmus, Bacillus kurstaki, Bacillus lacticola, Bacillus lactimorbus, Bacillus lactis, Bacillus laterosporus (also known as Brevibacillus laterosporus), Bacillus lautus, Bacillus lentimorbus, Bacillus lentus, Bacillus licheniformis, Bacillus maroccanus, Bacillus megaterium, Bacillus metiens, Bacillus mycoides, Bacillus natto, Bacillus nematocida, Bacillus nigrificans, Bacillus nigrum, Bacillus pantothenticus, Bacillus popillae, Bacillus psychrosaccharolyticus, Bacillus pumilus, Bacillus siamensis, Bacillus smithii, Bacillus sphaericus, Bacillus subtilis, Bacillus thuringiensis, Bacillus uniflagellatus, Bradyrhizobium japonicum, Brevibacillus brevis Brevibacillus laterosporus (formerly Bacillus laterosporus), Chromobacterium subtsugae, Delftia acidovorans, Herbaspirillum seropedicae, Herbaspirillum frisingense, Lactobacillus acidophilus, Lysobacter antibioticus, Lysobacter enzymogenes, Metakosakonia massiliensis, Metakosakonia intestinii, Paenibacillus alvei, Paenibacillus polymyxa, Paenibacillus popilliae (formerly Bacillus popilliae), Pantoea agglomerans, Paraburkholderia tropica, Paraburkholderia xenovorans, Pasteuria penetrans (formerly Bacillus penetrans), Pasteuria usgae, Pectobacterium carotovorum (formerly Erwinia carotovora), Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas benzenivorans, Pseudomonas cepacia (formerly known as Burkholderia cepacia). Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas proradix, Pseudomonas protegens, Pseudomonas putida, Pseudomonas syringae, Serratia entomophila, Serratia marcescens, Streptomyces colombiensis, Streptomyces galbus, Streptomyces goshikiensis, Streptomyces griseoviridis, Streptomyces lavendulae, Streptomyces prasinus, Streptomyces saracetictis, Streptomyces venezuelae, Xanthomonas campestris, Xenorhabdus luminescens, Xenorhabdus nematophila, Rhodococcus globerulus AQ719 (NRRL Accession No. B-21663), Bacillus sp. AQ175 (ATCC Accession No. 55608), Bacillus sp. AQ 177 (ATCC Accession No. 55609), Bacillus sp. AQ178 (ATCC Accession No. 53522), and Streptomyces sp. strain NRRL Accession No. B-30145. In some cases the bacterium may be Azotobacter chroococcum, Methanosarcina barkeri, Klesiella pneumoniae, Azotobacter vinelandii, Azospirillum brasilense, Rhodobacter spharoides, Rhodobacter capsulatus, Rhodobcter palustris, Rhodosporillum rubrum, Rhizobium leguminosarum or Rhizobium etli.

In some cases the bacterium may be a species of Clostridium, for example Clostridium pasteurianum, Clostridium beijerinckn, Clostridium perfringens, Clostridium tetani, Clostridium acetobutylicum.

In some cases, bacteria used with the methods and compositions of the present disclosure may be cyanobacteria. Examples of cyanobacterial genera include Anabaena (for example Anagaena sp. PCC7120), Nostoc (for example Nostoc punctiforme), or Synechocystis (for example Synechocystis sp. PCC6803).

In some cases, bacteria used with the methods and compositions of the present disclosure may belong to the phylum Chlorobi, for example Chlorobium tepidum.

In some cases, microbes used with the methods and compositions of the present disclosure may comprise a gene homologous to a known NifH gene. Sequences of known NifH genes may be found in, for example, the Zehr lab NifH database, (www.zehr.pmc.ucsc.edu/nifH_Database_Public/, Apr. 4, 2014), or the Buckley lab NifH database (www.css.cornell.edu/faculty/buckley/nifh.htm, and Gaby, John Christian, and Daniel H. Buckley. “A comprehensive aligned nifH gene database: a multipurpose tool for studies of nitrogen-fixing bacteria.” Database 2014 (2014): bau001.). In some cases, microbes used with the methods and compositions of the present disclosure may comprise a sequence which encodes a polypeptide with at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 96%, 98%, 99% or more than 99% sequence identity to a sequence from the Zehr lab NifH database, (www.zehr.pmc.ucsc.edu/nifH_Database_Public/, Apr. 4, 2014). In some cases, microbes used with the methods and compositions of the present disclosure may comprise a sequence which encodes a polypeptide with at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 96%, 98%, 99% or more than 99% sequence identity to a sequence from the Buckley lab NifH database, (Gaby, John Christian, and Daniel H. Buckley “A comprehensive aligned nifH gene database, a multipurpose tool for studies of nitrogen-fixing bacteria.” Database 2014 (2014): bau001.).

Microbes useful in the methods and compositions disclosed herein can be obtained by extracting microbes from surfaces or tissues of native plants; grinding seeds to isolate microbes; planting seeds in diverse soil samples and recovering microbes from tissues; or inoculating plants with exogenous microbes and determining which microbes appear in plant tissues. Non-limiting examples of plant tissues include a seed, seedling, leaf, cutting, plant, bulb, tuber, root, and rhizomes. In some cases, bacteria are isolated from a seed. The parameters for processing samples may be varied to isolate different types of associative microbes, such as rhizospheric, epiphytes, or endophytes. Bacteria may also be sourced from a repository, such as environmental strain collections, instead of initially isolating from a first plant. The microbes can be genotyped and phenotyped, via sequencing the genomes of isolated microbes, profiling the composition of communities in planta; characterizing the transcriptomic functionality of communities or isolated microbes; or screening microbial features using selective or phenotypic media (e.g., nitrogen fixation or phosphate solubilization phenotypes). Selected candidate strains or populations can be obtained via sequence data; phenotype data; plant data (e.g., genome, phenotype, and/or yield data); soil data (e.g., pH, N/P/K content, and/or bulk soil biotic communities); or any combination of these.

The bacteria and methods of producing bacteria described herein may apply to bacteria able to self-propagate efficiently on the leaf surface, root surface, or inside plant tissues without inducing a damaging plant defense reaction, or bacteria that are resistant to plant defense responses. The bacteria described herein may be isolated by culturing a plant tissue extract or leaf surface wash in a medium with no added nitrogen. However, the bacteria may be unculturable, that is, not known to be culturable or difficult to culture using standard methods known in the art. The bacteria described herein may be an endophyte or an epiphyte or a bacterium inhabiting the plant rhizosphere (rhizospheric bacteria). The bacteria obtained after repeating the steps of introducing genetic variation, exposure to a plurality of plants, and isolating bacteria from plants with an improved trait one or more times (e.g. 1, 2, 3, 4, 5, 10, 15, 25, or more times) may be endophytic, epiphytic, or rhizospheric. Endophytes are organisms that enter the interior of plants without causing disease symptoms or eliciting the formation of symbiotic structures, and are of agronomic interest because they can enhance plant growth and improve the nutrition of plants (e.g., through nitrogen fixation). The bacteria can be a seed-borne endophyte. Seed-borne endophytes include bacteria associated with or derived from the seed of a grass or plant, such as a seed-borne bacterial endophyte found in mature, dry, undamaged (e.g., no cracks, visible fungal infection, or prematurely germinated) seeds. The seed-borne bacterial endophyte can be associated with or derived from the surface of the seed; alternatively, or in addition, it can be associated with or derived from the interior seed compartment (e.g., of a surface-sterilized seed). In some cases, a seed-borne bacterial endophyte is capable of replicating within the plant tissue, for example, the interior of the seed. Also, in some cases, the seed-borne bacterial endophyte is capable of surviving desiccation.

The bacterial isolated according to methods of the disclosure, or used in methods or compositions of the disclosure, can comprise a plurality of different bacterial taxa in combination. By way of example, the bacteria may include Proteobacteria (such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Paraburkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azorhizobium, Azotobacter, Duganella, Delftia, Bradyrhizobium, Sinorhizobium, Metakosakonia and Halomonas), Firmicutes (such as Bacillus, Paenibacillus, Lactobacillus, Mycoplasma, and Acetabacterium), and Actinobacteria (such as Streptomyces, Rhodacoccus, Microbacterium, and Curtobacterium). The bacteria used in methods and compositions of this disclosure may include nitrogen fixing bacterial consortia of two or more species. In some cases, one or more bacterial species of the bacterial consortia may be capable of fixing nitrogen. In some cases, one or more species of the bacterial consortia may facilitate or enhance the ability of other bacteria to fix nitrogen. The bacteria which fix nitrogen and the bacteria which enhance the ability of other bacteria to fix nitrogen may be the same or different. In some examples, a bacterial strain may be able to fix nitrogen when in combination with a different bacterial strain, or in a certain bacterial consortia, but may be unable to fix nitrogen in a monoculture. Examples of bacterial genera which may be found in a nitrogen fixing bacterial consortia include, but are not limited to, Herbaspirillum, Azospirillum, Enterobacter, and Bacillus.

Bacteria that can be produced by the methods disclosed herein include Azotobacter sp., Bradyrhizobium sp., Klebsiella sp., and Sinorhizobium sp. In some cases, the bacteria may be selected from the group consisting of: Azotobacter vinelandii. Azospirillum brasilense, Bradyrhizobium japonicum, Klebsiella pneumoniae, and Sinorhizobium meliloti. In some cases, the bacteria may be of the genus Enterobacter or Rahnella. In some cases, the bacteria may be of the genus Frankia, or Clostridium. Examples of bacteria of the genus Clostridium include, but are not limited to, Clostridium acetobutilicum, Clostridium pasteurianum, Clostridium beijerinckii, Clostridium perfringens, and Clostridium tetani. In some cases, the bacteria may be of the genus Paenibacillus, for example Paenibacillus azotofixans, Paenibacillus borealis, Paenibacillus durus, Paenibacillus macerans, Paenibacillus polymyxa, Paenibacillus alvei, Paenibacillus amylolyticus, Paenibacillus campinasensis, Paenibacillus chibensis, Paenibacillus glucanolyticus, Paenibacillus illinoisensis, Paenibacillus larvae subsp. larvae, Paenibacillus larvae subsp. Pulvifaciens, Paenibacillus lautus, Paenibacillus macerans, Paenibacillus macquariensis, Paenibacillus macquariensis, Paenibacillus pabuli, Paenibacillus peoriae, or Paenibacillus polymyxa.

In some examples, bacteria isolated according to methods of the disclosure can be a member of one or more of the following taxa: Achromobacter, Acidithiobacillus, Acidovorax, Acidovoraz, Acinetobacter, Actinoplanes, Adlercreutzia, Aerococcus, Aeromonas, Afipia, Agromyces, Ancylobacter, Arthrobacter, Atopostipes, Azospirillum, Bacillus, Bdellovibrio, Beijerinckia, Bosea, Bradyrhizobium, Brevibacillus, Brevundimonas, Burkholderia, Candidatus Haloredivivus, Caulobacter, Cellulomonas, Cellvibrio, Chryseobacterium, Citrobacter, Clostridium, Coraliomargarita, Corynebacterium, Cupriavidus, Curtobacterium, Curvibacter, Deinococcus, Delftia, Desemzia, Devosia, Dokdonella, Dyella, Enhydrobacter, Enterobacter, Enterococcus, Erwinia, Escherichia, Escherichia Shigella, Exiguobacterium, Ferroglobus, Filimonas, Finegoldia, Flavisolibacter, Flavobacterium, Frigoribacterium, Gluconacetobacter, Hafnia, Halobaculum, Halomonas, Halosimplex, Herbaspirillum, Hymenobacter, Klebsiella, Kocuria, Kosakonia, Lactobacillus, Leclercia, Lentzea, Luteibacter, Luteimonas, Massilia, Mesorhizobium, Methylobacterium, Microbacterium, Micrococcus, Microvirga, Mycobacterium, Neisseria, Nocardia, Oceanibaculum, Ochrobactrum, Okibacterium, Oligotropha, Oryzihumus, Oxalophagus, Paenibacillus, Panteoa, Pantoea, Pelomonas, Perlucidibaca, Plantibacter, Polynucleobacter, Propionibacterium, Propioniciclava, Pseudoclavibacter, Pseudomonas, Pseudonocardia, Pseudoxonthomonas, Psychrobacter, Rahnella, Ralstonia, Rheinheimera, Rhizobium, Rhodococcus, Rhodopseudomonas, Roseateles, Ruminococcus, Sebaldella, Sediminibacillus, Sediminibacterium, Serratia, Shigella, Shinella, Sinorhizobium, Sinosporangium, Sphingobacterium, Sphingomonas, Sphingopyxis, Sphingosinicella, Staphylococcus, 25 Stenotrophomonas, Strenotrophomonas, Streptococcus, Streptomyces, Stygiolobus, Sulfurisphaera, Tatumella, Tepidimonas, Thermomonas, Thiobacillus, Variovorox, WPS-2 genera incertae sedis, Xanthomonas, and Zimmermannella.

In some cases, a bacterial species selected from at least one of the following genera are utilized: Enterobacter, Klebsiella, Kosakonia, and Rahnella. In some cases, a combination of bacterial species from the following genera are utilized: Enterobacter, Klebsiella, Kosakonia, and Rahnella. In some cases, the species utilized can be one or more of: Enterobacter sacchari, Klebsiella variicola, Kosakonia sacchari, and Rahnella aquatilis.

In some cases, a Gram positive microbe may have a Molybdenum-Iron nitrogenase system comprising: nifH, nifD, nifK, nifB, nifE, nifN, nifX, hesA, nifV, nifW, nifU, nifS, nifI1, and nifI2. In some cases, a Gram positive microbe may hove a vanadium nitrogenase system comprising: vnfDG, vnfK, vnfE, vnfN, vupC, vupB, vupA, vnfV, vnfR1, vnfH, vnfR2, vnfA (transcriptional regulator). In some cases, a Gram positive microbe may have an iron-only nitrogenase system comprising: anfK, anfG, anfD, anfH, anfA (transcriptional regulator). In some cases a Gram positive microbe may have a nitrogenase system comprising glnB, and glnK (nitrogen signaling proteins). Some examples of enzymes involved in nitrogen metabolism in Gram positive microbes include glnA (glutamine synthetase), gdh (glutamate dehydrogenase), bdh (3-hydroxybutyrate dehydrogenase), glutaminase, gltAB/gltB/gltS (glutamate synthase), asnA/asnB (aspartate-ammonia ligase/asparagine synthetase), and ansA/ansZ (asparaginase). Some examples of proteins involved in nitrogen transport in Gram positive microbes include amtB (ammonium transporter), glnK (regulator of ammonium transport), glnPHQ/glnQHMP (ATP-dependent glutamine/glutamate transporters), glnT/alsT/yrbD/yflA (glutamine-like proton symport transporters), and gltP/gltT/yhcl/nqt (glutamate-like proton symport transporters).

Examples of Gram positive microbes which may be of particular interest include Paenibacillus polymixa, Paenibacillus riograndensis, Paenibacillus sp., Frankia sp., Heliobacterium sp., Heliobacterium chlorum, Heliobacillus sp., Heliophilum sp., Heliorestis sp., Clostridium acetobutylicum, Clostridium sp., Mycobacterium flaum, Mycobacterium sp., Arthrobacter sp., Agromyces sp., Corynebacterium autitrophicum, Corynebacterium sp., Micromonspora sp., Propionibacteria sp., Streptomyces sp., and Microbacterium sp.

Some examples of genetic alterations which may be made in Gram positive microbes include, deleting glnR to remove negative regulation of BNF in the presence of environmental nitrogen, inserting different promoters directly upstream of the nif cluster to eliminate regulation by GlnR in response to environmental nitrogen, mutating glnA to reduce the rate of ammonium assimilation by the GS-GOGAT pathway, deleting amtB to reduce uptake of ammonium from the media, mutating glnA so it is constitutively in the feedback-inhibited (FBI-GS) state, to reduce ammonium assimilation by the GS-GOGAT pathway.

In some cases, glnR is the main regulator of N metabolism and fixation in Paenibacillus species. In some cases, the genome of a Paenibacillus species may not contain a gene to produce glnR In some cases, the genome of a Paenibacillus species may not contain a gene to produce glnE or glnD. In some cases, the genome of a Paenibacillus species may contain a gene to produce glnB or glnK. For example Paenibacillus sp. WLY78 doesn't contain a gene for glnB, or its homologs found in the archaeon Methanococcus maripaludis, nifI1 and nifI2. In some cases, the genomes of Paenibacillus species may be variable. For example, Paenibacillus polymixa E681 lacks glnK and gdh, has several nitrogen compound transporters, but only amtB appears to be controlled by GlnR In another example, Paenibacillus sp. JDR2 has glnK, gdh and most other central nitrogen metabolism genes, has many fewer nitrogen compound transporters, but does have glnPHQ controlled by GlnR. Paenibacillus riograndensis SBR5 contains a standard glnRA operon, an fdx gene, a main nif operon, a secondary nif operon, and an anf operon (encoding iron-only nitrogenase). Putative glnR/tnrA sites were found upstream of each of these operons. GlnR may regulate all of the above operons, except the anf operon. GlnR may bind to each of these regulatory sequences as a dimer.

Paenibacillus N-fixing strains may fall into two subgroups: Subgroup I, which contains only a minimal nif gene cluster and subgroup n, which contains a minimal cluster, plus an uncharacterized gene between nifX and hesA, and often other clusters duplicating some of the nif genes, such as nifH, nifHDK, nifBEN, or clusters encoding vanadium nitrogenase (vnf) or iron-only nitrogenase (anf) genes.

In some cases, the genome of a Paenibacillus species may not contain a gene to produce glnB or glnK. In some cases, the genome of a Paenibacillus species may contain a minimal nif cluster with 9 genes transcribed from a sigma-70 promoter. In some cases a Paenibacillus nif cluster may be negatively regulated by nitrogen or oxygen. In some cases, the genome of a Paenibacillus species may not contain a gene to produce sigma-54. For example, Paenibacillus sp. WLY78 does not contain a gene for sigma-54. In some cases, a nif cluster may be regulated by glnR, and/or TnrA. In some cases, activity of a nif cluster may be altered by altering activity of glnR, and/or TnrA.

In Bacilli, glutamine synthetase (GS) is feedback-inhibited by high concentrations of intracellular glutamine, causing a shift in confirmation (referred to as FBI-GS). Nif clusters contain distinct binding sites for the regulators GlnR and TnrA in several Bacilli species. GlnR binds and represses gene expression in the presence of excess intracellular glutamine and AMP. A role of GlnR may be to prevent the influx and intracellular production of glutamine and ammonium under conditions of high nitrogen availability. TnrA may bind and/or activate (or repress) gene expression in the presence of limiting intracellular glutamine, and/or in the presence of FBI-GS. In some cases the activity of a Bacilli nif cluster may be altered by altering the activity of GlnR.

Feedback-inhibited glutamine synthetase (FBI-GS) may bind GlnR and stabilize binding of GlnR to recognition sequences. Several bacterial species have a GlnR/TnrA binding site upstream of the nif cluster. Altering the binding of FBI-GS and GlnR may alter the activity of the nif pathway.

Sources of Microbes

The bacteria (or any microbe according to the disclosure) may be obtained from any general terrestrial environment, including its soils, plants, fungi, animals (including invertebrates) and other biota, including the sediments, water and biota of lakes and rivers; from the marine environment, its biota and sediments (for example, sea water, marine muds, marine plants, marine invertebrates (for example, sponges), marine vertebrates (for example, fish)); the terrestrial and marine geosphere (regolith and rock, for example, crushed subterranean rocks, sand and clays); the cryosphere and its meltwater; the atmosphere (for example, filtered aerial dusts, cloud and rain droplets); urban, industrial and other man-made environments (for example, accumulated organic and mineral matter on concrete, roadside gutters, roof surfaces, and road surfaces).

The plants from which the bacteria (or any microbe according to the disclosure) are obtained may be a plant having one or more desirable traits, for example a plant which naturally grows in a particular environment or under certain conditions of interest. By way of example, a certain plant may naturally grow in sandy soil or sand of high salinity, or under extreme temperatures, or with little water, or it may be resistant to certain pests or disease present in the environment, and it may be desirable for a commercial crop to be grown in such conditions, particularly if they are, for example, the only conditions available in a particular geographic location. By way of further example, the bacteria may be collected from commercial crops grown in such environments, or more specifically from individual crop plants best displaying a trait of interest amongst a crop grown in any specific environment; for example the fastest-growing plants amongst a crop grown in saline-limiting soils, or the least damaged plants in crops exposed to severe insect damage or disease epidemic, or plants having desired quantities of certain metabolites and other compounds, including fiber content, oil content, and the like, or plants displaying desirable colors, taste or smell. The bacteria may be collected from a plant of interest or any material occurring in the environment of interest, including fungi and other animal and plant biota, soil, water, sediments, and other elements of the environment as referred to previously.

The bacteria (or any microbe according to the disclosure) may be isolated from plant tissue. This isolation can occur from any appropriate tissue in the plant, including for example root, stem and leaves, and plant reproductive tissues. By way of example, conventional methods for isolation from plants typically include the sterile excision of the plant material of interest (e.g. root or stem lengths, leaves), surface sterilization with an appropriate solution (e.g. 2% sodium hypochlorite), after which the plant material is placed on nutrient medium for microbial growth. Alternatively, the surface-sterilized plant material can be crushed in a sterile liquid (usually water) and the liquid suspension, including small pieces of the crushed plant material spread over the surface of a suitable solid agar medium, or media, which may or may not be selective (e.g. contain only phytic acid as a source of phosphorus). This approach is especially useful for bacteria which form isolated colonies and can be picked off individually to separate plates of nutrient medium, and further purified to a single species by well-known methods. Alternatively, the plant root or foliage samples may not be surface sterilized but only washed gently thus including surface-dwelling epiphytic microorganisms in the isolation process, or the epiphytic microbes can be isolated separately, by imprinting and lifting off pieces of plant roots, stem or leaves onto the surface of an agar medium and then isolating individual colonies as above. This approach is especially useful for bacteria, for example. Alternatively, the roots may be processed without washing off small quantities of soil attached to the roots, thus including microbes that colonize the plant rhizosphere. Otherwise, soil adhering to the roots can be removed, diluted and spread out onto agar of suitable selective and non-selective media to isolate individual colonies of rhizospheric bacteria.

Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedures

The microbial deposits of the present disclosure were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure (Budapest Treaty).

Applicants state that pursuant to 37 C.F.R. § 1.808(a)(2) “all restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably removed upon the granting of the patent” This statement is subject to paragraph (b) of this section (i.e. 37 C.F.R. § 1.808(b)).

The Enterobacter sacchari has now been reclassified as Kosakonia sacchari, the name for the organism may be used interchangeably throughout the manuscript.

Many microbes of the present disclosure are derived from two wild-type strains. Strain CI006 is a bacterial species previously classified in the genus Enterobacter (see aforementioned reclassification into Kosakonia). Strain 0019 is a bacterial species classified in the genus Rahnella. The deposit information for the 0006 Kosakonia wild type (WT) and 0019 Rahnella WT are found in the below Table 1.

Some microorganisms described in this application were deposited on Jan. 6, 2017 or Aug. 11, 2017 with the Bigelow National Center for Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Me. 04544, USA. As aforementioned, all deposits were made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The Bigelow National Center for Marine Algae and Microbiota accession numbers and dates of deposit for the aforementioned Budapest Treaty deposits are provided in Table 1.

Biologically pure cultures of Kosakonia sacchari (WT), Rahnella aquatilis (WT), and a variant/remodeled Kosakonia sacchari strain were deposited on Jan. 6, 2017 with the Bigelow National Center for Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Me. 04544, USA, and assigned NCMA Patent Deposit Designation numbers 201701001, 201701003, and 201701002, respectively. The applicable deposit information is found below in Table 1.

Biologically pure cultures of variant/remodeled Kosakonia sacchari strains were deposited on Aug. 11, 2017 with the Bigelow National Center for Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Me. 04544, USA, and assigned NCMA Patent Deposit Designation numbers 201708004, 201708003, and 201708002, respectively. The applicable deposit information is found below in Table 1.

A biologically pure culture of Klebsiella variicola (WT) was deposited on Aug. 11, 2017 with the Bigelow National Center for Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Me. 04544, USA, and assigned NCMA Patent Deposit Designation number 201708001. Biologically pure cultures of two Klebsiella variicola variants/remodeled strains were deposited on Dec. 20, 2017 with the Bigelow National Center for Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Me. 04544, USA, and assigned NCMA Patent Deposit Designation numbers 201712001 and 201712002, respectively. The applicable deposit information is found below in Table 1.

Biologically pure cultures of two Kosakonia sacchari variants/remodeled strains were deposited on Dec. 23, 2019 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA and assigned ATCC Patent Deposit Numbers PTA-126575 and PTA-126576. Biologically pure cultures of four Klebsiella variicola variants/remodeled strains were deposited on Dec. 23, 2019 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA and assigned ATCC Patent Deposit Numbers PTA-126577, PTA-126578, PTA-126579 and PTA-126580. A biologically pure culture of a Paenibacillus polymyxa (WT) strain was deposited on Dec. 23, 2019 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA and assigned ATCC Patent Deposit Number PTA-126581. A biologically pure culture of a Paraburkholderia tropica (WT) strain was deposited on Dec. 23, 2019 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA and assigned ATCC Patent Deposit Number PTA-126582. A biologically pure culture of a Herbaspirillum aquaticum (WT) strain was deposited on Dec. 23, 2019 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA and assigned ATCC Patent Deposit Number PTA-126583. Biologically pure cultures of four Metakosakonia intestini variants/remodeled strains were deposited on Dec. 23, 2019 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA and assigned ATCC Patent Deposit Numbers PTA-126584, PTA-126586, PTA-126587 and PTA-126588. A biologically pure culture of a Metakosakonia intestini (WT) strain was deposited on Dec. 23, 2019 with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA and assigned ATCC Patent Deposit Number PTA-126585. The applicable deposit information is found below in Table 1.

TABLE 1 Microorganisms Deposited under the Budapest Treaty Pivot Strain Designation (some strains have multiple Accession Date of Depository designations) Taxonomy Number Deposit NCMA CI006, Kosakonia sacchari (WT) 201701001 Jan. 6, 2017 PBC6.1, 6 NCMA CI019, 19 Rahnella aquatilis (WT) 201701003 Jan. 6, 2017 NCMA CM029, 6-412 Kosakonia sacchari 201701002 Jan. 6, 2017 NCMA 6-403 CM037 Kosakonia sacchari 201708004 Aug. 11, 2017 NCMA 6-404, CM38, Kosakonia sacchari 201708003 Aug. 11, 2017 PBC6.38 NCMA CM094, 6-881, Kosakonia sacchari 201708002 Aug. 11, 2017 PBC6.94 NCMA CI137, 137, Klebsiella variicola (WT) 201708001 Aug. 11, 2017 PB137 NCMA 137-1034 Klebsiella variicola 201712001 Dec. 20, 2017 NCMA 137-1036 Klebsiella variicola 201712002 Dec. 20, 2017 ATCC 6-2425 Kosakonia sacchari PTA-126575 Dec. 23, 2019 ATCC 6-2634 Kosakonia sacchari PTA-126576 Dec. 23, 2019 ATCC 137-1968 Klebsiella variicola PTA-126577 Dec. 23, 2019 ATCC 137-2219 Klebsiella variicola PTA-126578 Dec. 23, 2019 ATCC 137-2237 Klebsiella variicola PTA-126579 Dec. 23, 2019 ATCC 137-2285 Klebsiella variicola PTA-126580 Dec. 23, 2019 ATCC 41 Paenibacillus polymyxa (WT) PTA-126581 Dec. 23, 2019 ATCC 8 Paraburkholderia tropica (WT) PTA-126582 Dec. 23, 2019 ATCC 3069 Herbaspirillum aquaticum (WT) PTA-126583 Dec. 23, 2019 ATCC 910-3655 Metakosakonia intestini PTA-126584 Dec. 23, 2019 ATCC 910 Metakosakonia intestini (WT) PTA-126585 Dec. 23, 2019 ATCC 910-3963 Metakosakonia intestini PTA-126586 Dec. 23, 2019 ATCC 910-3961 Metakosakonia intestini PTA-126587 Dec. 23, 2019 ATCC 910-3994 Metakosakonia intestini PTA-126588 Dec. 23, 2019

Isolated and Biologically Pure Microorganisms

The present disclosure, in certain embodiments, provides isolated and biologically pure microorganisms that have applications, inter alia, in agriculture. The disclosed microorganisms can be utilized in their isolated and biologically pure states, as well as being formulated into compositions (see below section for exemplary composition descriptions). Furthermore, the disclosure provides microbial compositions containing at least two members of the disclosed isolated and biologically pure microorganisms, as well as methods of utilizing said microbial compositions. Furthermore, the disclosure provides for methods of modulating nitrogen fixation in plants via the utilization of the disclosed isolated and biologically pure microbes.

In some aspects, the isolated and biologically pure microorganisms of the disclosure are those from Table 1. In other aspects, the isolated and biologically pure microorganisms of the disclosure are derived from a microorganism of Table 1. For example, a strain, child, mutant, or derivative, of a microorganism from Table 1 are provided herein. The disclosure contemplates all possible combinations of microbes listed in Table 1, said combinations sometimes forming a microbial consortia. The microbes from Table 1, either individually or in any combination, can be combined with any plant, active molecule (synthetic, organic, etc.), adjuvant, carrier, supplement, or biological, mentioned in the disclosure.

In some aspects, the disclosure provides microbial compositions comprising species as grouped in Tables 2-8. In some aspects, these compositions comprising various microbial species are termed a microbial consortia or consortium.

With respect to Tables 2-8, the letters A through I represent a non-limiting selection of microorganisms of the present disclosure, defined as:

A=Microbe with accession number 201701001 identified in Table 1;

B=Microbe with accession number 201701003 identified in Table 1;

C=Microbe with accession number 201701002 identified in Table 1;

D=Microbe with accession number 201708004 identified in Table 1;

E=Microbe with accession number 201708003 identified in Table 1;

F=Microbe with accession number 201708002 identified in Table 1;

G=Microbe with accession number 201708001 identified in Table 1;

H=Microbe with accession number 201712001 identified in Table 1; and

I=Microbe with accession number 201712002 identified in Table 1.

TABLE 2 Eight and Nine Strain Compositions A, B, C, D, E, F, G, H A, B, C, D, E, F, G, I A, B, C, D, E, F, H, I A, B, C, D, E, G, H, I A, B, C, D, F, G, H, I A, B, C, E, F, G, H, I A, B, D, E, F, G, H, I A, C, D, E, F, G, H, I B, C, D, E, F, G, H, I A, B, C, D, E, F, G, H, I

TABLE 3 Seven Strain Compositions A, B, C, D, E, F, G A, B, C, D, E, F, H A, B, C, D, E, F, I A, B, C, D, E, G, H A, B, C, D, E, G, I A, B, C, D, E, H, I A, B, C, D, F, G, H A, B, C, D, F, G, I A, B, C, D, F, H, I A, B, C, D, G, H, I A, B, C, E, F, G, H A, B, C, E, F, G, I A, B, C, E, F, H, I A, B, C, E, G, H, I A, B, C, F, G, H, I A, B, D, E, F, G, H A, B, D, E, F, G, I A, B, D, E, F, H, I A, B, D, E, G, H, I A, B, D, F, G, H, I A, B, E, F, G, H, I A, C, D, E, F, G, H A, C, D, E, F, G, I A, C, D, E, F, H, I A, C, D, E, G, H, I A, C, D, F, G, H, I A, C, E, F, G, H, I A, D, E, F, G, H, I B, C, D, E, F, G, H B, C, D, E, F, G, I B, C, D, E, F, H, I B, C, D, E, G, H, I B, C, D, F, G, H, I B, C, E, F, G, H, I B, D, E, F, G, H, I C, D, E, F, G, H, I

TABLE 4 Six Strain Compositions A, B, C, D, E, F A, B, C, D, E, G A, B, C, D, E, H A, B, C, D, E, I A, B, C, D, F, G A, B, C, D, F, H A, B, C, D, F, I A, B, C, D, G, H A, B, C, D, G, I A, B, C, D, H, I A, B, C, E, F, G A, B, C, E, F, H A, B, C, E, F, I A, B, C, E, G, H A, B, C, E, G, I A, B, C, E, H, I A, B, C, F, G, H A, B, C, F, G, I A, B, C, F, H, I A, B, C, G, H, I A, B, D, E, F, G A, B, D, E, F, H A, B, D, E, F, I A, B, D, E, G, H A, B, D, E, G, I A, B, D, E, H, I A, B, D, F, G, H A, B, D, F, G, I D, E, F, G, H, I C, E, F, G, H, I A, B, D, F, H, I A, B, D, G, H, I A, B, E, F, G, H A, B, E, F, G, I A, B, E, F, H, I A, B, E, G, H, I A, B, F, G, H, I A, C, D, E, F, G A, C, D, E, F, H A, C, D, E, F, I A, C, D, E, G, H A, C, D, E, G, I A, C, D, E, H, I A, C, D, F, G, H A, C, D, F, G, I A, C, D, F, H, I A, C, D, G, H, I A, C, E, F, G, H A, C, E, F, G, I A, C, E, F, H, I A, C, E, G, H, I A, C, F, G, H, I A, D, E, F, G, H A, D, E, F, G, I A, D, E, F, H, I A, D, E, G, H, I A, D, F, G, H, I A, E, F, G, H, I B, C, D, E, F, G B, C, D, E, F, H B, C, D, E, F, I B, C, D, E, G, H B, C, D, E, G, I B, C, D, E, H, I B, C, D, F, G, H B, C, D, F, G, I B, C, D, F, H, I B, C, D, G, H, I B, C, E, F,G, H B, C, E, F, G, I B, C, E, F, H, I B, C, E, G, H, I B, C, F, G, H, I B, D, E, F, G, H B, D, E, F, G, I B, D, E, F, H, I B, D, E, G, H, I B, D, F, G, H, I B, E, F, G, H, I C, D, E, F, G, H C, D, E, F, G, I C, D, E, F, H, I C, D, E, G, H, I C, D, F, G, H, I

TABLE 5 Five Strain Compositions A, B, C, D, E A, B, C, D, F A, B, C, D, G A, B, C, D, H A, B, C, D, I A, B, C, E, F A, B, C, E, G A, B, C, E, H A, B, C, F, H A, B, C, F, G A, B, C, F, I A, B, C, G, H A, B, C, G, I A, B, C, H, I A, B, D, E, F A, B, D, E, G A, B, D, E, I A, B, D, F, G A, B, D, F, H A, B, D, F, I A, B, D, G, H A, B, D, G, I A, B, D, H, I A, B, E, F, G A, B, E, F, I A, B, E, G, H A, B, E, G, I A, B, E, H, I A, B, F, G, H A, B, F, G, I A, B, F, H, I A, B, G, H, I A, C, D, E, G A, C, D, E, H A, C, D, E, I A, C, D, F, G A, C, D, F, H A, C, D, F, I A, C, D, G, H A, C, D, G, I A, C, E, F, G A, C, E, F, H A, C, E, F, I A, C, E, G, H A, C, E, G, I A, C, E, H, I A, C, F, G, H A, C, F, G, I A, C, G, H, I A, D, E, F, G A, D, E, F, H A, D, E, F, I A, D, E, G, H A, D, E, G, I A, D, E, H, I A, D, F, G, H A, D, F, H, I A, D, G, H, I A, E, F, G, H A, E, F, G, I A, E, F, H, I A, E, G, H, I A, F, G, H, I B, C, D, E, F B, C, D, E, H B, C, D, E, I B, C, D, F, G B, C, D, F, H B, C, D, F, I B, C, D, G, H B, C, D, G, I B, C, D, H, I B, C, E, F, H B, C, E, F, I B, C, E, G, H B, C, E, G, I B, C, E, H, I B, C, F, G, H B, C, F, G, I B, C, F, H, I B, D, E, F, G B, D, E, F, H B, D, E, F, I B, D, E, G, H B, D, E, G, I B, D, E, H, I B, D, F, G, H B, D, F, G, I B, D, G, H, I B, E, F, G, H B, E, F, G, I B, E, F, H, I B, E, G, H, I B, F, G, H, I C, D, E, F, G C, D, E, F, H C, D, E, G, H C, D, E, G, I C, D, E, H, I C, D, F, G, H C, D, F, G, I C, D, F, H, I C, D, G, H, I C, E, F, G, H C, E, F, H, I C, E, G, H, I C, F, G, H, I D, E, F, G, H D, E, F, G, I D, E, F, H, I D, E, G, H, I D, F, G, H, I A, B, C, E, I A, B, D, E, H A, B, E, F, H A, C, D, E, F A, C, D, H, I A, C, F, H, I A, D, F, G, I B, C, D, E, G B, C, E, F, G B, C, G, H, I B, D, F, H, I C, D, E, F, I C, E, F, G, I E, F, G, H, I

TABLE 6 Four Strain Compositions A, B, C, D A, B, C, E A, B, C, F A, B, C, G A, B, C, H A, B, C, I A, B, D, E A, B, D, F D, G, H, I A, B, D, G A, B, D, H A, B, D, I A, B, E, F A, B, E, G A, B, E, H A, B, E, I A, B, F, G E, F, G, H A, B, F, H A, D, F, H A, D, F, I A, D, G, H A, D, G, I A, D, H, I A, E, F, G A, E, F, H E, F, G, I A, B, F, I A, B, G, H A, B, G, I A, B, H, I A, C, D, E A, C, D, F A, C, D, G A, C, D, H E, F, H, I A, C, D, I A, C, E, F A, C, E, G A, C, E, H A, C, E, I A, C, F, G A, C, F, H A, C, F, I E, G, H, I A, C, G, H A, C, G, I A, C, H, I A, D, E, F A, D, E, G A, D, E, H A, D, E, I A, D, F, G F, G, H, I A, E, F, I A, E, G, H A, E, G, I A, E, H, I A, F, G, H A, F, G, I A, F, H, I A, G, H, I D, E, F, H B, C, D, E B, C, D, F B, C, D, G B, C, D, H B, C, D, I B, C, E, F B, C, E, G B, C, E, H D, E, F, I B, C, E, I B, C, F, G B, C, F, H B, C, F, I B, C, G, H B, C, G, I B, C, H, I B, D, E, F D, E, G, H B, D, E, G B, D, E, H B, D, E, I B, D, F, G B, D, F, H B, D, F, I B, D, G, H B, D, G, I D, E, G, I B, D, H, I B, E, F, G B, E, F, H B, E, F, I B, E, G, H B, E, G, I B, E, H, I B, F, G, H D, E, H, I B, F, G, I B, F, H, I B, G, H, I C, D, E, F C, D, E, G C, D, E, H C, D, E, I C, D, F, G D, F, G, H C, D, F, H C, D, F, I C, D, G, H C, D, G, I C, D, H, I C, E, F, G C, E, F, H C, E, F, I D, F, G, I C, E, G, H C, E, G, I C, E, H, I C, F, G, H C, F, G, I C, F, H, I C, G, H, I D, E, F, G D, F, H, I

TABLE 7 Three Strain Compositions A, B, C A, B, D A, B, E A, B, F A, B, G A, B, H A, B, I A, C, D A, C, E G, H, I E, F, H A, C, F A, C, G A, C, H A, C, I A, D, E A, D, F A, D, G A, D, H A, D, I F, H, I E, F, G A, E, F A, E, G A, E, H A, E, I A, F, G A, F, H A, F, I A, G, H A, G, I F, G, I D, H, I A, H, I B, C, D B, C, E B, C, F B, C, G B, C, H B, C, I B, D, E B, D, F F, G, H D, G, I B, D, G B, D, H B, D, I B, E, F B, E, G B, E, H B, E, I B, F, G B, F, H E, H, I E, F, I B, F, I B, G, H B, G, I B, H, I C, D, E C, D, F C, D, G C, D, H C, D, I E, G, I D, G, H C, E, F C, E, G C, E, H C, E, I C, F, G C, F, H C, F, I C, G, H C, G, I E, G, H D, F, I C, H, I D, E, F D, E, G D, E, H D, E, I D, F, G D, F, H

TABLE 8 Two Strain Compositions A, B A, C A, D A, E A, F A, G A, H A, I B, C B, D B, E B, F B, G B, H B, I C, D C, E C, F C, G C, H C, I D, E D, F D, G D, H D, I E, F E, G E, H E, I F, G F, H F, I G, H G, I H, I

In some embodiments, microbial compositions may be selected from any member group from Tables 2-8.

In some embodiments, any microbe of the present disclosure may be modified or optimized to excrete ammonium constitutively or non-constitutively. In some embodiments, the modification of any microbe of the present disclosure is a transgenic modification. In some embodiments, the microbes are already a transgenic organism and the strains are modified such that they no longer contain a transgenic element. In some embodiments, the modification of any microbe of the present disclosure is a non-transgenic modification. In some embodiments, any two or more PGPR are combined in a microbial consortia. In some embodiments, any two or more microbes of the present disclosure are combined in a microbial consortia. In some embodiments, the microbial consortia are applied to any one or more plants of the present disclosure and/or the surrounding soil or growth medium. In some embodiments, any PGPR is applied to any one or more of the plants of the present disclosure and/or the surrounding soil or growth medium.

In some embodiments, the microbes of the present disclosure are modified or optimized to enhance or increase the ability to colonize plants. In some embodiments, the enhanced or increased ability to colonize plants is an enhanced or increased ability to colonize the surface of the roots.

Agricultural Compositions

Compositions comprising bacteria or bacterial populations produced according to methods described herein and/or having characteristics as described herein can be in the form of a liquid, a foam, or a dry product. Compositions comprising bacteria or bacterial populations produced according to methods described herein and/or having characteristics as described herein may also be used to improve plant traits. In some examples, a composition comprising bacterial populations may be in the form of a dry powder, a slurry of powder and water, or a flowable seed treatment. The compositions comprising bacterial populations may be coated on a surface of a seed, and may be in liquid form.

The composition can be fabricated in bioreactors such as continuous stirred tank reactors, batch reactors, and on the farm. In some examples, compositions can be stored in a container, such as a jug or in mini bulk. In some examples, compositions may be stored within an object selected from the group consisting of a bottle, jar, ampule, package, vessel, bag, box, bin, envelope, carton, container, silo, shipping container, truck bed, and/or case.

Compositions may also be used to improve plant traits. In some examples, one or more compositions may be coated onto a seed. In some examples, one or more compositions may be coated onto a seedling. In some examples, one or more compositions may be coated onto a surface of a seed. In some examples, one or more compositions may be coated as a layer above a surface of a seed. In some examples, a composition that is coated onto a seed may be in liquid form, in dry product form, in foam form, in a form of a slurry of powder and water, or in a flowable seed treatment. In some examples, one or more compositions may be applied to a seed and/or seedling by spraying, immersing, coating, encapsulating, and/or dusting the seed and/or seedling with the one or more compositions. In some examples, multiple bacteria or bacterial populations can be coated onto a seed and/or a seedling of the plant. In some examples, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more than ten bacteria of a bacterial combination can be selected from one of the following genera. Acidovorax, Agrobacterium, Bacillus, Burkholderia, Chryseobacterium, Curtobacterium, Enterobacter, Escherichia, Methylobacterium, Paenibacillus, Pantoea, Pseudomonas, Ralstonia, Saccharibacillus, Sphingomonas, and Stenotrophomonas.

In some examples, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more than ten bacteria and bacterial populations of an endophytic combination are selected from one of the following families: Baciliaceae, Burkholderiaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae, Methylobacteriaceae, Microbacteriaceae, Paenibacillileae, Pseudomonnaceae, Rhizobiaceae, Sphingomonadaceae, Xanthomonadaceae, Cladosporiaceae, Gnomoniaceae, Incertae sedis, Ixtsiosphaeriaceae, Netriaceae, and Pleosporaceae.

In some examples, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least night, at least ten, or more than ten bacteria and bacterial populations of an endophytic combination are selected from one of the following families: Baciliaceae, Burkholderiaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae, Methylobacteriaceae, Microbacteriaceae, Paenibacillileae, Pseudomonnaceae, Rhizobiaceae, Sphingomonadaceae, Xanthomonadaceae, Cladosporiaceae, Gnomoniaceae, Incertae sedis, Lasiosphaeriaceae, Netriaceae, Pleosporaceae.

Examples of compositions may include seed coatings for commercially important agricultural crops, for example, sorghum, canola, tomato, strawberry, barley, rice, maize, and wheat. Examples of compositions can also include seed coatings for corn, soybean, canola, sorghum, potato, rice, vegetables, cereals, and oilseeds. Seeds as provided herein can be genetically modified organisms (GMO), non-GMO, organic, or conventional. In some examples, compositions may be sprayed on the plant aerial parts, or applied to the roots by inserting into furrows in which the plant seeds are planted, watering to the soil, or dipping the roots in a suspension of the composition. In some examples, compositions may be dehydrated in a suitable manner that maintains cell viability and the ability to artificially inoculate and colonize host plants. The bacterial species may be present in compositions at a concentration of between 10⁸ to 10¹⁰ CFU/ml. In some examples, compositions may be supplemented with trace metal ions, such as molybdenum ions, iron ions, manganese ions, or combinations of these ions. The concentration of ions in examples of compositions as described herein may between about 0.1 mM and about 50 mM. Some examples of compositions may also be formulated with a carrier, such as beta-glucan, carboxylmethyl cellulose (CMC), bacterial extracellular polymeric substance (EPS), sugar, animal milk, or other suitable carriers. In some examples, peat or planting materials can be used as a carrier, or biopolymers in which a composition is entrapped in the biopolymer can be used as a carrier. The compositions comprising the bacterial populations described herein can improve plant traits, such as promoting plant growth, maintaining high chlorophyll content in leaves, increasing fruit or seed numbers, and increasing fruit or seed unit weight.

The compositions comprising the bacterial populations described herein may be coated onto the surface of a seed. As such, compositions comprising a seed coated with one or more bacteria described herein are also contemplated. The seed coating can be formed by mixing the bacterial population with a porous, chemically inert granular carrier. Alternatively, the compositions may be inserted directly into the furrows into which the seed is planted or sprayed onto the plant leaves or applied by dipping the roots into a suspension of the composition. An effective amount of the composition can be used to populate the sub-soil region adjacent to the roots of the plant with viable bacterial growth, or populate the leaves of the plant with viable bacterial growth. In general, an effective amount is an amount sufficient to result in plants with improved traits (e.g. a desired level of nitrogen fixation).

Bacterial compositions described herein can be formulated using an agriculturally acceptable carrier. The formulation useful for these embodiments may include at least one member selected from the group consisting of a tackifier, a microbial stabilizer, a fungicide, an antibacterial agent, a preservative, a stabilizer, a surfactant, an anti-complex agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a fertilizer, a rodenticide, a desiccant, a bactericide, a nutrient, or any combination thereof. In some examples, compositions may be shelf-stable. For example, any of the compositions described herein can include an agriculturally acceptable carrier (e.g., one or more of a fertilizer such as a non-naturally occurring fertilizer, an adhesion agent such as a non-naturally occurring adhesion agent, and a pesticide such as a non-naturally occurring pesticide). A non-naturally occurring adhesion agent can be, for example, a polymer, copolymer, or synthetic wax. For example, any of the coated seeds, seedlings, or plants described herein can contain such an agriculturally acceptable carrier in the seed coating. In any of the compositions or methods described herein, an agriculturally acceptable carrier can be or can include a non-naturally occurring compound (e.g., a non-naturally occurring fertilizer, a non-naturally occurring adhesion agent such as a polymer, copolymer, or synthetic wax, or a non-naturally occurring pesticide). Non-limiting examples of agriculturally acceptable carriers are described below. Additional examples of agriculturally acceptable carriers are known in the art.

In some cases, bacteria are mixed with an agriculturally acceptable carrier. The carrier can be a solid carrier or liquid carrier, and in various forms including microspheres, powders, emulsions and the like. The carrier may be any one or more of a number of carriers that confer a variety of properties, such as increased stability, wettability, or dispersability. Wetting agents such as natural or synthetic surfactants, which can be nonionic or ionic surfactants, or a combination thereof can be included in the composition. Water-in-oil emulsions can also be used to formulate a composition that includes the isolated bacteria (see, for example, U.S. Pat. No. 7,485,451). Suitable formulations that may be prepared include wettable powders, granules, gels, agar strips or pellets, thickeners, and the like, microencapsulated particles, and the like, liquids such as aqueous flowables, aqueous suspensions, water-in-oil emulsions, etc. The formulation may include grain or legume products, for example, ground grain or beans, broth or flour derived from grain or beans, starch, sugar, or oil.

In some embodiments, the agricultural carrier may be soil or a plant growth medium. Other agricultural carriers that may be used include water, fertilizers, plant-based oils, humectants, or combinations thereof. Alternatively, the agricultural carrier may be a solid, such as diatomaceous earth, loam, silica, alginate, clay, bentonite, vermiculite, seed cases, other plant and animal products, or combinations, including granules, pellets, or suspensions. Mixtures of any of the aforementioned ingredients are also contemplated as carriers, such as but not limited to, pesta (flour and kaolin clay), agar or flour-based pellets in loam, sand, or clay, etc. Formulations may include food sources for the bacteria, such as barley, rice, or other biological materials such as seed, plant parts, sugar cane bagasse, hulls or stalks from grain processing, ground plant material or wood from building site refuse, sawdust or small fibers from recycling of paper, fabric, or wood.

For example, a fertilizer can be used to help promote the growth or provide nutrients to a seed, seedling, or plant. Non-limiting examples of fertilizers include nitrogen, phosphorous, potassium, calcium, sulfur, magnesium, boron, chloride, manganese, iron, zinc, copper, molybdenum, and selenium (or a salt thereof). Additional examples of fertilizers include one or more amino acids, salts, carbohydrates, vitamins, glucose, NaCl, yeast extract, NH₄H₂PO₄, (NH₄)₂SO₄, glycerol, valine, L-leucine, lactic acid, propionic acid, succinic acid, malic acid, citric acid, KH tartrate, xylose, lyxose, and lecithin. In one embodiment the formulation can include a tackifier or adherent (referred to as an adhesive agent) to help bind other active agents to a substance (e.g., a surface of a seed). Such agents are useful for combining bacteria with carriers that can contain other compounds (e.g., control agents that are not biologic), to yield a coating composition. Such compositions help create coatings around the plant or seed to maintain contact between the microbe and other agents with the plant or plant part. In one embodiment, adhesives are selected from the group consisting of: alginate, gums, starches, lecithins, formononetin, polyvinyl alcohol, alkali formononetinate, hesperetin, polyvinyl acetate, cephalins, Gum Arabic, Xanthan Gum, Mineral Oil, Polyethylene Glycol (PEG), Polyvinyl pyrrolidone (PVT), Arabino-galactan, Methyl Cellulose, PEG 400, Chitosan, Polyacrylamide, Polyacrylate, Polyacrylonitrile, Glycerol, Triethylene glycol, Vinyl Acetate, Gellan Gum, Polystyrene, Polyvinyl, Carboxymethyl cellulose, Gum Ghatti, and polyoxyethylene-polyoxybutylene block copolymers.

In some embodiments, the adhesives can be, e.g. a wax such as carnauba wax, beeswax, Chinese wax, shellac wax, spermaceti wax, candelilla wax, castor wax, ouricury wax, and rice bran wax, a polysaccharide (e.g., starch, dextrins, maltodextrins, alginate, and chitosans), a fat, oil, a protein (e.g., gelatin and zeins), gum arables, and shellacs. Adhesive agents can be non-naturally occurring compounds, e.g., polymers, copolymers, and waxes. For example, non-limiting examples of polymers that can be used as an adhesive agent include: polyvinyl acetates, polyvinyl acetate copolymers, ethylene vinyl acetate (EVA) copolymers, polyvinyl alcohols, polyvinyl alcohol copolymers, celluloses (e.g., ethylcelluloses, methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses, and carboxymethylcelluloses), polyvinylpyrolidones, vinyl chloride, vinylidene chloride copolymers, calcium lignosulfonates, acrylic copolymers, polyvinylacrylates, polyethylene oxide, acylamide polymers and copolymers, polyhydroxyethyl acrylate, methylacrylamide monomers, and polychloroprene.

In some examples, one or more of the adhesion agents, anti-fungal agents, growth regulation agents, and pesticides (e.g., insecticide) are non-naturally occurring compounds (e.g., in any combination). Additional examples of agriculturally acceptable carriers include dispersants (e.g., polyvinylpyrrolidone/vinyl acetate PVPIVA S-630), surfactants, binders, and filler agents.

The formulation can also contain a surfactant. Non-limiting examples of surfactants include nitrogen-surfactant blends such as Prefer 28 (Cenex), Surf-N (US), Inhance (Brandt), P-28 (Wilfarm) and Patrol (Helena); esterified seed oils include Sun-It II (AmCy), MSO (UAP), Scoil (Agsco), Hasten (Wilfarm) and Mes-100 (Drexel); and organo-silicone surfactants include Silwet L77 (UAP), Silikin (Terra), Dyne-Amic (Helena), Kinetic (Helena), Sylgard 309 (Wilbur-Ellis) and Century (Precision). In one embodiment, the surfactant is present at a concentration of between 0.01% v/v to 10% v/v. In another embodiment, the surfactant is present at a concentration of between 0.1% v/v to 1% v/v.

In certain cases, the formulation includes a microbial stabilizer. Such an agent can include a desiccant, which can include any compound or mixture of compounds that can be classified as a desiccant regardless of whether the compound or compounds are used in such concentrations that they in fact have a desiccating effect on a liquid inoculant Such desiccants are ideally compatible with the bacterial population used, and should promote the ability of the microbial population to survive application on the seeds and to survive desiccation. Examples of suitable desiccants include one or more of trehalose, sucrose, glycerol, and Methylene glycol. Other suitable desiccants include, but are not limited to, non-reducing sugars and sugar alcohols (e.g., mannitol or sorbitol). The amount of desiccant introduced into the formulation can range from about 5% to about 50% by weight/volume, for example, between about 10% to about 40%, between about 15% to about 35%, or between about 20% to about 30%. In some cases, it is advantageous for the formulation to contain agents such as a fungicide, an antibacterial agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, bactericide, or a nutrient. In some examples, agents may include protectants that provide protection against seed surface-borne pathogens. In some examples, protectants may provide some level of control of soil-borne pathogens. In some examples, protectants may be effective predominantly on a seed surface.

In some examples, a fungicide may include a compound or agent, whether chemical or biological, that can inhibit the growth of a fungus or kill a fungus. In some examples, a fungicide may include compounds that may be fungistatic or fungicidal. In some examples, fungicide can be a protectant, or agents that are effective predominantly on the seed surface, providing protection against seed surface-borne pathogens and providing some level of control of soil-borne pathogens. Non-limiting examples of protectant fungicides include captan, maneb, thiram, or fludioxonil.

In some examples, fungicide can be a systemic fungicide, which can be absorbed into the emerging seedling and inhibit or kill the fungus inside host plant tissues. Systemic fungicides used for seed treatment include, but are not limited to the following, azoxystrobin, carboxin, mefenoxam, metalaxyl, thiabendazole, trifloxystrobin, and various triazole fungicides, including difenoconazole, ipconazole, tebuconazole, and triticonazole. Mefenoxam and metalaxyl are primarily used to target the water mold fungi Pythium and Phytophthora. Some fungicides are preferred over others, depending on the plant species, either because of subtle differences in sensitivity of the pathogenic fungal species, or because of the differences in the fungicide distribution or sensitivity of the plants. In some examples, fungicide can be a biological control agent, such as a bacterium or fungus. Such organisms may be parasitic to the pathogenic fungi, or secrete toxins or other substances which can kill or otherwise prevent the growth of fungi. Any type of fungicide, particularly ones that are commonly used on plants, can be used as a control agent in a seed composition.

In some examples, the seed coating composition comprises a control agent which has antibacterial properties. In one embodiment, the control agent with antibacterial properties is selected from the compounds described herein elsewhere. In another embodiment, the compound is Streptomycin, oxytetracycline, oxolinic acid, or gentamicin. Other examples of antibacterial compounds which can be used as part of a seed coating composition include those based on dichlorophene and benzylalcohol hemi formal (Proxel® from ICI or Acticide® RS from Thor Chemie and Kathon® MK 25 from Rohm & Haas) and isothiazolinone derivatives such as alkylisothiazolinones and benzisothiazolinones (Acticide® MBS from Thor Chemie).

In some examples, growth regulator is selected from the group consisting of: Abscisic acid, amidochlor, ancymidol, 6-benzylaminopurine, brassinolide, butralin, chlormequat (chlormequat chloride), choline chloride, cyclanilide, daminozide, dikegulac, dimethipin, 2,6-dimethylpuridine, ethephon, flumetralin, flurprimidol, fluthiacet, forchlorfenuron, gibberellic acid, inabenfide, indole-3-acetic acid, maleic hydrazide, mefluidide, mepiquat (mepiquat chloride), naphthaleneacetic acid, N-6-benzyladenine, paclobutrazol, prohexadione phosphorotrithioate, 2,3,5-tri-iodobenzoic acid, trinexapac-ethyl and uniconazole. Additional non-limiting examples of growth regulators include brassinosteroids, cytokinines (e.g., kinetin and zeatin), auxins (e.g., indolylacetic acid and indolylacetyl aspartate), flavonoids and isoflavanoids (e.g., formononetin and diosmetin), phytoaixins (e.g., glyceolline), and phytoalexin-inducing oligosaccharides (e.g., pectin, chitin, chitosan, polygalacuronic acid, and oligogalacturonic acid), and gibellerins. Such agents are ideally compatible with the agricultural seed or seedling onto which the formulation is applied (e.g., it should not be deleterious to the growth or health of the plant). Furthermore, the agent is ideally one which does not cause safety concerns for human, animal or industrial use (e.g., no safety issues, or the compound is sufficiently labile that the commodity plant product derived from the plant contains negligible amounts of the compound).

Some examples of nematode-antagonistic biocontrol agents include ARF18; 30 Arthrobotrys spp.; Chaetomium spp.; Cylindrocarpon spp., Exophilia spp.; Fusarium spp.; Gliocladium spp., Hirsutella spp., Lecanicillium spp., Monacrosporium spp., Myrothecium spp.; Neocosmospora spp., Paecilomyces spp.; Pochonia spp., Stagonospora spp.; vesicular-arbuscular mycorrhizal fungi, Burkholderia spp.; Pasteuria spp., Brevibacillus spp., Pseudomonas spp.; and Rhizobacteria. Particularly preferred nematode-antagonistic biocontrol agents include ARF18, Arthrobotrys oligospora, Arthrobotrys dactyloides, Chaetomium globosum, Cylindrocarpon heteronema, Exophilia jeanselmei, Exophilia pisciphila, Fusarium aspergilus, Fusarium solani, Gliocladium catenulatum, Gliocladium roseum, Gliocladium vixens, Hirsutella rhossiliensis, Hirsutella minnesotensis, Lecanicillium lecanii, Monacrosporium drechsleri, Monacrosporium gephyropagum, Myrotehcium verrucaria, Neocosmospora vasinfecta, Paecilomyces lilacinus, Pochonia chlamydosporia, Stagonospora heteroderae, Stagonospora phaseoli, vesicular-arbuscular mycorrhizal fungi, Burkholderia cepacia, Pasteuria penetrans, Pasteuria thornei, Pasteuria nishizawae, Pasteuria ramosa, Pastrueia usage, Brevibacillus laterosporus strain G4, Pseudomonas fluorescens and Rhizobacteria.

Some examples of nutrients can be selected from the group consisting of a nitrogen fertilizer including, but not limited to Urea, Ammonium nitrate, Ammonium sulfate, Non-pressure nitrogen solutions, Aqua ammonia, Anhydrous ammonia, Ammonium thiosulfate, Sulfur-coated urea, Urea-formaldehydes, IBDU, Polymer-coated urea. Calcium nitrate, Ureaform, and Methylene urea, phosphorous fertilizers such as Diammonium phosphate, Monoammonium phosphate, Ammonium polyphosphate. Concentrated superphosphate and Triple superphosphate, and potassium fertilizers such as Potassium chloride, Potassium sulfate, Potassium-magnesium sulfate. Potassium nitrate. Such compositions can exist as free salts or ions within the seed coat composition. Alternatively, nutrients/fertilizers can be complexed or chelated to provide sustained release over time.

Some examples of rodenticides may include selected from the group of substances consisting of 2-isovalerylindan-1,3-dione, 4-(quinoxalin-2-ylamino) benzenesulfonamide, alpha-chlorohydrin, aluminum phosphide, antu, arsenous oxide, barium carbonate, bisthiosemi, brodifacoum, bromadiolone, bromethalin, calcium cyanide, chloralose, chlorophacinone, cholecalciferol, coumachlor, coumafuryl, coumatetralyl, crimidine, difenacoum, difethialone, diphacinone, ergocalciferol, flocoumafen, fluoroacetamide, flupropadine, flupropadine hydrochloride, hydrogen cyanide, iodomethane, lindane, magnesium phosphide, methyl bromide, norbormide, phosacetim, phosphine, phosphorus, pindone, potassium arsenite, pyrinuron, scilliroside, sodium arsenite, sodium cyanide, sodium fluoroacetate, strychnine, thallium sulfate, warfarin and zinc phosphide.

In the liquid form, for example, solutions or suspensions, bacterial populations can be mixed or suspended in water or in aqueous solutions. Suitable liquid diluents or carriers include water, aqueous solutions, petroleum distillates, or other liquid carriers.

Solid compositions can be prepared by dispersing the bacterial populations in and on an appropriately divided solid carrier, such as peat, wheat, bran, vermiculite, clay, talc, bentonite, diatomaceous earth, fuller's earth, pasteurized soil, and the like. When such formulations are used as wettable powders, biologically compatible dispersing agents such as non-ionic, anionic, amphoteric, or cationic dispersing and emulsifying agents can be used.

The solid carriers used upon formulation include, for example, mineral carriers such as kaolin clay, pyrophyllite, bentonite, montmorillonite, diatomaceous earth, acid white soil, vermiculite, and pearlite, and inorganic salts such as ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, ammonium chloride, and calcium carbonate. Also, organic fine powders such as wheat flour, wheat bran, and rice bran may be used. The liquid carriers include vegetable oils such as soybean oil and cottonseed oil, glycerol, ethylene glycol, polyethylene glycol, propylene glycol, polypropylene glycol, etc.

Pests

Agricultural compositions of the disclosure, which may comprise any microbe taught herein, are sometimes combined with one or more pesticides.

The pesticides that are combined with the microbes of the disclosure may target any of the pests mentioned below.

“Pest” includes but is not limited to, insects, fungi, bacteria, nematodes, mites, ticks and the like. Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Lepidoptera and Coleoptera.

Those skilled in the art will recognize that not all compounds are equally effective against all pests. Compounds that may be combined with microbes of the disclosure may display activity against insect pests, which may include economically important agronomic, forest, greenhouse, nursery ornamentals, food and fiber, public and animal health, domestic and commercial structure, household and stored product pests.

As aforementioned, the agricultural compositions of the disclosure (which may comprise any microbe taught herein) are in embodiments combined with one or more pesticides. These pesticides may be active against any of the following pests:

Larvae of the order Lepidoptera include, but are not limited to, armyworms, cutworms, loopers and heliothines in the family Noctuidae Spodoptera frugiperda J E Smith (fall army worm); S. exigua Hubner (beet armyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar); Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus (cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogonia Morrison (western cutworm); A. subterranea Fabricius (granulate cutworm); Alabama argillacea Hubner (cotton leaf worm); Trichoplusia ni Hubner (cabbage looper); Pseudoplusia includens Walker (soybean looper); Anticarsia gemmatalis Hubner (velvet bean caterpillar); Hypena scabra Fabricius (green clover worm); Heliothis virescens Fabricius (tobacco budworm); Pseudaletia unipuncta Haworth (armyworm); Athens mindara Barnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris (darksided cutworm); Earias insulana Boisduval (spiny bollworm); E. vittella Fabricius (spotted bollworm); Helicoverpa armigera Hubner (American bollworm); H. zea Boddie (corn earworm or cotton bollworm); Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialis Grote (citrus cutworm); borers, case bearers, webworms, coneworms, and skeletonizers from the family Pyralidae Ostrinia nubilalis Hubner (European corn borer); Amyelois transitella Walker (naval orangeworm); Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautella Walker (almond moth); Chilo suppressalis Walker (rice stem borer); C. partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth); Crambus caliginosellus Clemens (corn root webworm); C. teterrellus Zincken (bluegrass webworm); Cnaphalocrocis medinalis Guenee (rice leaf roller); Desmia fimeralis Hubner (grape leaffolder); Diaphania hyalinata Linnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraea grandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius (surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestia elutella Hubner (tobacco (cacao) moth); Galleria mellonella Linnaeus (greater wax moth), Herpetogramma licarsisalis Walker (sod webworm); Homoeosoma eiectellum Hulst (sunflower moth); Elasmopalpus lignosellus Zeller (lesser cornstalk borer); Achroia griseila Fabricius (lesser wax moth), Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalis Walker (tea tree web moth); Maruca testulalis Geyer (bean pod borer); Plodia interpunctella Hubner (Indian meal moth); Scirpophaga incertulas Walker (yellow stem borer); Udea rubigalis Guenee (celery leaftier), and leafrollers, budworms, seed worms and fruit worms in the family Tortricidae Acleris gloverana Walsingham (Western blackheaded budworm); A. variana Fernald (Eastern blackheaded budworm); Archips argyrospila Walker (fruit tree leaf roller); A. rosana Linnaeus (European leaf roller); and other Archips species, Adoxophyes orana Fischer von Rosslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham (banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C. pomonella Linnaeus (colding moth); Platynota flavedana Clemens (variegated leafroller); P. stultana Walsingham (omnivorous leafroller); Lobesia botrana Denis & Schiffermuller (European grape vine moth); Spilonota ocellana Denis & Schiffermuller (eyespotted bud moth); Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguella Hubner (vine moth); Bonagota salubricola Meyrick (Brazilian apple leafroller); Grapholita molesta Busck (oriental fruit moth); Suleima helianthana Riley (sunflower bud moth); Argyrotaenia spp.; Choristoneura spp.

Selected other agronomic pests in the order Lepidoptera include, but are not limited to, Alsophila pometaria Harris (fall cankerworm); Anarsia lineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith (orange striped oakworm); Antheraea pernyi Guerin-Meneville (Chinese Oak Tussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiella Busck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfa caterpillar); Datana integerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomos subsignaria Hubner (elm spanworm); Erannis tiliaria Harris (linden looper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisina americana Guerin-Meneville (grapeleaf skeletonizer); Hemileuca oliviae Cockrell (range caterpillar); Hyphantria cunea Drury (fall web-worm); Keiferia lycopersicella Walsingham (tomato pinworm); Lambdina fiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellaria lugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus (satin moth); Lymantria dispar Linnaeus (gypsy moth); Manduca quinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M. sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumata Linnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm), Papilio cresphontes Cramer (giant swallowtail orange dog); Phryganidia californica Packard (California oakworm), Phyllocnistis citrella Stainton (citrus leafminer); Phyllonorycter blancardella Fabricius (spotted tentiform leafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapae Linnaeus (small white butterfly), P. napi Linnaeus (green veined white butterfly); Platyptilia carduidactyla Riley (artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth); Pectinophora gossypiella Saunders (pink bollworm); Pontia protodice Boisduval and Leconte (Southern cabbage-worm); Sabulodes aegrotata Guenee (omnivorous looper); Schizura concinna J. E. Smith (red humped caterpillar); Sitotroga cerealella Olivier (Angoumois grain moth); Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar); Tineola bisselliella Hummel (webbing clothes moth); Tuta absoluta Meyrick (tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothis subflexa Guenee; Malacosoma spp. and Orgyia spp.; Ostrinia nubilalis (European corn borer); seed corn maggot; Agrotis ipsilon (black cutworm).

Larvae and adults of the order Coleoptera including weevils from the families Anthribidae, Bruchidae and Curculionidae (including, but not limited to: Anthonomus grandis Boheman (boll weevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil); Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (rice weevil); Hypera punctata Fabricius (clover leaf weevil); Cylindrocopturus adspersus LeConte (sunflower stem weevil); Smicronyx fulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (gray sunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug)); flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetles and leafminers in the family Chrysomelidae (including, but not limited to: Leptinotarsa decemlineata Say (Colorado potato beetle); Diabrotica virgifera virgifera LeConte (western corn rootworm); D. barberi Smith and Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber (southern corn rootworm); Chaetocnema pulicaria Melsheimer (corn flea beetle); Phyllotreta cruciferae Goeze (Crucifer flea beetle); Phyllotreta striolata (stripped flea beetle); Colaspis brunnea Fabricius (grape colaspis); Oulema melanopus Linnaeus (cereal leaf beetle); Zygogramma exclamationis Fabricius (sunflower beetle)); beetles from the family Coccinellidae (including, but not limited to; Epilachna varivestis Mulsant (Mexican bean beetle)); chafers and other beetles from the family Scarabaeidae (including, but not limited to: Popillia japonica Newman (Japanese beetle); Cyclocephala borealis Arrow (northern masked chafer, white grub); C. immaculata Olivier (southern masked chafer, white grub); Rhizotrogus majalis Razoumowsky (European chafer); Phyllophaga crinita Burmeister (white grub), Ligyrus gibbosus De Geer (carrot beetle)); carpet beetles from the family Dermestidae; wireworms from the family Elateridae, Eleodes spp., Melanotus spp.; Conoderus spp.; Limonius spp., Agriotes spp.; Ctenicera spp.; Aeolus spp.; bark beetles from the family Scolytidae and beetles from the family Tenebrionidae; Cerotoma trifurcate (bean leaf beetle); and wireworm.

Adults and immatures of the order Diptera, including leafminers Agromyza parvicornis Loew (corn blotch leafminer), midges (including, but not limited to: Contarinia sorghicola Coquillett (sorghum midge); Mayetiola destructor Say (Hessian fly); Sitodiplosis mosellana Gehin (wheat midge); Neolasioptera murtfeldtiana Felt, (sunflower seed midge)); fruit flies (Tephritidae), Oscinella frit Linnaeus (fruit flies); maggots (including, but not limited to: Delia platura Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulb fly) and other Delia spp., Meromyza americana Fitch (wheat stem maggot); Musca domestica Linnaeus (house flies); Fannia canicularis Linnaeus, F. femoralis Stein (lesser house flies); Stomoxys calcitrans Linnaeus (stable flies)); face flies, horn flies, blow flies, Chrysomya spp.; Phormia spp. and other muscoid fly pests, horse flies Tabanus spp.; bot flies Gastrophilus spp.; Oestrus spp.; cattle grubs Hypoderma spp.; deer flies Chrysops spp.; Melophagus ovinus Linnaeus (keds) and other Brachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; black flies Prosimulium spp.; Simulium spp.; biting midges, sand flies, sciarids, and other Nematocera.

Adults and nymphs of the orders Hemiptera and Homoptera such as, but not limited to, adelgids from the family Adelgidae, plant bugs from the family Miridae, cicadas from the family Cicadidae, leafhoppers, Empoasca spp.; from the family Cicadellidae, planthoppers from the families Cixiidae, Flatidae, Fulgoroidea, Issidae and Delphacidae, treehoppers from the family Membracidae, psyllids from the family Psyllidae, whiteflies from the family Aleyrodidae, aphids from the family Aphididae, phylloxera from the family Phylloxeridae, mealybugs from the family Pseudococcidae, scales from the families Asterolecanidae, Coccidae, Dactylopiidae, Diaspididae, Eriococcidae Ortheziidae, Phoenicococcidae and Margarodidae, lace bugs from the family Tingidae, stink bugs from the family Pentatomidae, cinch bugs, Blissus spp.; and other seed bugs from the family Lygaeidae, spittlebugs from the family Cercopidae squash bugs from the family Coreidae and red bugs and cotton stainers from the family Pyrrhocoridae.

Agronomically important members from the order Homoptera further include, but are not limited to: Acyrthisiphon pisum Harris (pea aphid), Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black bean aphid), A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicis Forbes (corn root aphid), A. pomi De Geer (apple aphid); A. spiraecola Patch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid); Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxia Kurdjumov/Mordvilko (Russian wheat aphid); Dysaphis plantaginea Paaserini (rosy apple aphid); Eriosoma lanigerum Hausmann (woolly apple aphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopterus pruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnip aphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphum euphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach potato aphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid); Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch (corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphis graminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcane aphid); Sitobion avenae Fabricius (English grain aphid); Therioaphis maculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer de Fonscolombe (black citrus aphid) and T. citricida Kirkaldy (brown citrus aphid); Melanaphis sacchari (sugarcane aphid); Adelges spp. (adelgids); Phylloxera devastatrix Pergande (pecan phylloxera); Bemisia tabaci Gennadius (tobacco whitefly, sweetpotato whitefly); B. argentifolii Bellows & Perring (silverleaf whitefly); Dialeurodes citri Ashmead (citrus whitefly); Trialeurodes abutiloneus (bandedwinged whitefly) and T. vaporariorum Westwood (greenhouse whitefly); Empoasca fabae Harris (potato leafhopper); Laodelphax striatellus Fallen (smaller brown planthopper); Macrolestes quadrilineatus Forbes (aster leafhopper); Nephotettix cinticeps Uhler (green leafhopper); N. nigropictus Stal (rice leafhopper); Nilaparvata lugens Stal (brown planthopper); Peregrinus maidis Ashmead (corn planthopper); Sogatella furcifera Horvath (white backed planthopper); Sogatodes orizicola Muir (rice delphacid); Typhlocyba pomaria McAtee (white apple leafhopper); Erythroneoura spp. (grape leafhoppers); Magicicada septendecim Linnaeus (periodical cicada); Icerya purchasi Maskell (cottony cushion scale); Quadraspidiotus perniciosus Cornstock (San Jose scale); Planococcus citri Risso (citrus mealybug); Pseudococcus spp. (other mealybug complex); Cacopsylla pyricola Foerster (pear psylla), Trioza diospyri Ashmead (persimmon psylla).

Species from the order Hemiptera include, but are not limited to; Acrosternum hilare Say (green stink bug); Anasa tristis De Geer (squash bug); Blissus leucopterus leucopterus Say (chinch bug), Corythuca gossypii Fabricius (cotton lace bug); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellus Herrich-Schaffer (cotton stainer); Euschistus servus Say (brown stink bug); E. variolarius Palisot de Beauvais (one spotted stink bug); Graptostethus spp. (complex of seed bugs), Leptoglossus corculus Say (leaf footed pine seed bug); Lygus lineolaris Palisot de Beauvais (tarnished plant bug); L. hesperus Knight (Western tarnished plant bug); L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius (European tarnished plant bug); Lygocoris pabulinus Linnaeus (common green capsid); Nezara viridula Linnaeus (southern green stink bug); Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas (large milk-weed bug); Pseudatomoscelis seriatus Reuter (cotton flea hopper).

Hemiptera such as, Calocoris norvegicus Gmelin (strawberry bug); Orthops campestris Linnaeus; Plesiocoris rugicollis Fallen (apple capsid); Cyrtopeltis modestus Distant (tomato bug); Cyrtopeltis notatus Distant (suckfly); Spanagonictis albofasciatus Reuter (whitemarked fleahopper); Diaphnocoris chlorionis Say (honeylocust plant bug); Labopidicola allii Knight (onion plant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper); Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatus Fabricius (four lined plant bug); Nysius ericae Schilling (false chinch bug); Nysius raphanus Howard (false chinch bug); Nezara viridula Linnaeus (Southern green stink bug); Eurygaster spp; Coreidae spp.; Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidae spp. and Cimicidae spp.

Adults and larvae of the order Acari (mites) such as Aceria tosichella Keifer (wheat curl mite); Petrobia latens Muller (brown wheat mite); spider mites and red mites in the family Tetranychidae, Panonychus ulmi Koch (European red mite); Tetranychus urticae Koch (two spotted spider mite); (T. mcdanieli McGregor (McDaniel mite); T. cirmabarimis Boisduval (carmine spider mite); T. turkestani Ugarov & Nikolski (strawberry spider mite); flat mites in the family Tenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust and bud mites in the family Eriophyidae and other foliar feeding mites and mites important in human and animal health, i.e., dust mites in the family Epidermoptidae, follicle mites in the family Demodicidae, grain mites in the family Glycyphagidae, ticks in the order Ixodidae. Ixodes scapularis Say (deer tick); I. holocyclus Neumann (Australian paralysis tick); Dermacentor variabilis Say (American dog tick); Amblyomma americanum Linnaeus (lone star tick) and scab and itch mites in the families Psoroptidae, Pyemotidae and Sarcoptidae.

Insect pests of the order Thysanura, such as Lepisma saccharina Linnaeus (silverfish); Thermobia domestica Packard (firebrat).

Additional arthropod pests include: spiders in the order Araneae such as Loxosceles reclusa Gertsch and Mulaik (brown recluse spider) and the Latrodectus mactans Fabricius (black widow spider) and centipedes in the order Scutigeromorpha such as Scutigera coleoptrata Linnaeus (house centipede).

Superfamily of stink bugs and other related insects including but not limited to species belonging to the family Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorus guildini, Enschistus servus, Acrostemum hilare, Euschistus heros, Euschistus tristigmus, Acrostemum hilare, Dichelops furcatus, Dichelops melacanthus, and Bagrada hilaris (Bagrada Bug)), the family Plataspidae (Megacopta cribraria-Bean plataspid) and the family Cydnidae (Scaptocoris castanea-Root stink bug) and Lepidoptera species including but not limited to: diamond-back moth, e.g., Helicoverpa zea Boddie; soybean looper, e.g., Pseudoplusia includens Walker and velvet bean caterpillar e.g., Anticarsia gemmatalis Hubner.

Nematodes include parasitic nematodes such as root-knot, cyst and lesion nematodes, including Heterodera spp., Meloidogyne spp. and Globodera spp.; particularly members of the cyst nematodes, including, but not limited to, Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode) and Globodera rostochiensis and Globodera pailida (potato cyst nematodes). Lesion nematodes include Pratylenchus spp.

Pesticidal Compositions Comprising a Pesticide and Microbe of the Disclosure

As aforementioned, agricultural compositions of the disclosure, which may comprise any microbe taught herein, are sometimes combined with one or more pesticides. Pesticides can include herbicides, insecticides, fungicides, nematicides, etc.

In some embodiments the pesticides/microbial combinations can be applied in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with other compounds. These compounds can be fertilizers, weed killers, cryoprotectants, surfactants, detergents, pesticidal soaps, dormant oils, polymers, and/or time release or biodegradable carrier formulations that permit long term dosing of a target area following a single application of the formulation. They can also be selective herbicides, chemical insecticides, virucides, microbicides, amoebicides, pesticides, fungicides, bacteriocides, nematicides, molluscicides or mixtures of several of these preparations, if desired, together with further agriculturally acceptable carriers, surfactants or application promoting adjuvants customarily employed in the art of formulation. Suitable carriers (i.e. agriculturally acceptable carriers) and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, sticking agents, tackifiers, binders or fertilizers. Likewise the formulations may be prepared into edible baits or fashioned into pest traps to permit feeding or ingestion by a target pest of the pesticidal formulation.

Exemplary chemical compositions, which may be combined with the microbes of the disclosure, include:

Fruits/Vegetables Herbicides: Atrazine, Bromacil, Diuron, Glyphosate, Linuron, Metribuzin, Simazine, Trifluralin, Fluazifop, Glufosinate, Halo sulfuron Gowan, Paraquat, Propyzamide, Sethoxydim, Butafenacil, Halosulfuron, Indaziflam; Fruits/Vegetables Insecticides: Aldicarb, Bacillus thuringiensis, Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin, Diazinon, Malathion, Abamectin, Cyfluthrin/betacyfluthrin, Esfenvalerate, Lambda-cyhalothrin, Acequinocyl, Bifenazate, Methoxyfenozide, Novaluron, Chromafenozide, Thiacloprid, Dinotefuran, FluaCrypyrim, Tolfenpyrad, Clothianidin, Spirodiclofen, Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr, Cyazypyr, Spinoteram, Triflumuron, Spirotetramat, Imidacloprid, Flubendiamide, Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumetofen, Cyanopyrafen, Imidacloprid, Clothianidin, Thiamethoxam, Spinotoram, Thiodicarb, Flonicamid, Methiocarb, Emamectin benzoate, Indoxacarb, Forthiazate, Fenamiphos, Cadusaphos, Pyriproxifen, Fenbutatin oxide, Hexthiazox, Methomyl, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on; Fruits Vegetables Fungicides: Carbendazim, Chlorothalonil, EBDCs, Sulphur, Thiophanate-methyl, Azoxystrobin, Cymoxanil, Fluazinam, Fosetyl, Iprodione, Kresoxim-methyl, Metalaxyl/mefenoxam, Trifloxystrobin, Ethaboxam, Iprovalicarb, Trifloxystrobin, Fenhexamid, Oxpoconazole fumarate, Cyazofamid, Fenamidone, Zoxamide, Picoxystrobin, Pyraclostrobin, Cyflufenamid, Boscalid;

Cereals Herbicides: Isoproturon, Bromoxynil, Ioxynil, Phenoxies, Chlorsulfuron, Clodinafop, Diclofop, Diflufenican, Fenoxaprop, Florasulam, Fluoroxypyr, Metsulfuron, Triasulfuron, Flucarbazone, Iodosulfuron, Propoxycarbazone, Picolin-afen, Mesosulfuron, Beflubutamid, Pinoxaden, Amidosulfuron, Thifensulfuron Methyl, Tribenuron, Flupyrsulfuron, Sulfosulfuron, Pyrasulfotole, Pyroxsulam, Flufenacet, Tralkoxydim, Pyroxasulfon; Cereals Fungicides: Carbendazim, Chlorothalonil, Azoxystrobin, Cyproconazole, Cyprodinil, Fenpropimorph, Epoxiconazole, Kresoxim-methyl, Quinoxyfen, Tebuconazole, Trifloxystrobin, Simeconazole, Picoxystrobin, Pyraclostrobin, Dimoxystrobin, Prothioconazole, Fluoxastrobin; Cereals Insecticides: Dimethoate, Lambda-cyhalothrin, Deltamethrin, alpha-Cypermethrin, β-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dmetofuran, Clorphyriphos, Metamidophos, Oxidemethon methyl, Pirimicarb, Methiocarb;

Maize Herbicides: Atrazine, Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, S-Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole, S-Metolachlor, Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione, Foramsulfuron, Topramezone, Tembotrione, Saflufenacil, Thiencarbazone, Flufenacet, Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyrifos, Bifenthrin, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin, Terbufos, Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide, Triflumuron, Rynaxypyr, Deltamethrin, Thiodicarb, β-Cyfluthrin, Cypermethrin, Bifenthrin, Lufenuron, Triflumoron, Tefluthrin, Tebupirim-phos, Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid, Dinetofuran, Avermectin, Methiocarb, Spirodiclofen, Spirotetramat; Maize Fungicides: Fenitropan, Thiram, Prothioconazole, Tebuconazole, Trifloxystrobin;

Rice Herbicides: Butachlor, Propanil, Azimsulfuron, Bensulfuron, Cyhalo-fop, Daimuron, Fentrazamide, Imazosulfuron, Mefenacet, Oxaziclomefone, Pyrazosulfuron, Pyributicarb, Quinclorac, Thiobencarb, Indanofan, Flufenacet, Fentrazamide, Halosulfuron, Oxaziclomefone, Benzobicyclon, Pyriftalid, Penoxsulam, Bispyribac, Oxadiargyl, Ethoxysulfuron, Pretilachlor, Mesotrione, Tefuryltrione, Oxadiazone, Fenoxaprop, Pyrimisulfan; Rice Insecticides: Diazinon, Fenitro-thion, Fenobucarb, Monocrotophos, Benfuracarb, Buprofezin, Dinotefuran, Fipronil, Imidacloprid, Isoprocarb, Thiacloprid, Chromafenozide, Thiacloprid, Dinotefuran, Clothianidin, Ethiprole, Flubendiamide, Rynaxypyr, Deltamethrin, Acetamiprid, Thiamethoxam, Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Cypermethrin, Chlorpyriphos, Cartap, Methamidophos, Etofen-prox, Triazophos, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Carbofuran, Benfuracarb; Rice Fungicides: Thiophanate-methyl, Azoxystrobin, Carpropamid, Edifenphos, Ferimzone, Iprobenfos, Isoprothiolane, Pencycuron, Probenazole, Pyroquilon, Tricyclazole, Trifloxystrobin, Diclocymet, Fenoxanil, Simeconazole, Tiadinil;

Cotton Herbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn, Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate, Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron, Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; Cotton Insecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin, Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid, Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin, Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl, Flonicamid, Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin, Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran, Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl) methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen, Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton Fungicides: Etridiazole, Metalaxyl, Quintozene;

Soybean Herbicides: Alachlor, Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl, Fenoxaprop, Fomesafen, Flu-azifop, Glyphosate, Imazamox, Imazaquin, Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim, Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl, Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr, Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole, Deltamethrin, β-Cyfluthrin, gamma and lambda Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl] (2,2-difluorethyl)amino]furan-2(5H)-on, Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb, beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Cyproconazole, Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole, Trifloxystrobin, Prothioconazole, Tetraconazole;

Sugarbeet Herbicides: Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate, Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim, Triflusulfuron, Tepral-oxydim, Quizalofop; Sugarbeet Insecticides: Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Deltamethrin, β-Cyfluthrin, gamma/lambda Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluor-ethyl)amino]furan-2(5H)-on, Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran;

Canola Herbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate, Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop, Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin, Carbendazim, Fludioxonil, Iprodione, Prochloraz, Vinclozolin; Canola Insecticides: Carbofuran organophos-phates, Pyrethroids, Thiacloprid, Deltamethrin, Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dineto-furan, β-Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole, Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr, 4-[[(6-Chlorpyridin-3-yl)methyl] (2,2-difluorethyl)amino] furan-2(5H)-on.

Insecticidal Compositions Comprising an Insecticide and Microbe of the Disclosure

As aforementioned, agricultural compositions of the disclosure, which may comprise any microbe taught herein, are sometimes combined with one or more insecticides. Exemplary insecticides which may be combined with microbes of the disclosure can be found in Table 9. Exemplary pesticides which may be combined with microbes of the disclosure can be found in Table 10.

In some embodiments, insecticidal compositions may be included in the compositions set forth herein, and can be applied to a plants) or a part(s) thereof simultaneously or in succession, with other compounds. Insecticides include ammonium carbonate, aqueous potassium silicate, boric acid, copper sulfate, elemental sulfur, lime sulfur, sucrose octanoate esters, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, abamectin, notenone, fenazaquin, fenpyroximate, pyridaben, pyrimedifen, tebufenpyrad, tolfenpyrad, acephate, emamectin benzoate, lepimectin, milbemectin, hdroprene, kinoprene, methoprene, fenoxycarb, pyriproxyfen, methryl bromide and other alkyl halides, fulfuryl fluoride, chloropicrin, borax, disodium octaborate, sodium borate, sodium metaborate, tartar emetic, dazomet, metam, pymetrozine, pyrifluquinazon, flofentezine, diflovidazin, hexythiazox, bifenazate, thiamethoxam, imidacloprid, fenpyroximate, azadirachtin, permethrin, esfenvalerate, acetamiprid, bifenthrin, indoxacarb, azadirachtin, pyrethrin, imidacloprid, beta-cyfluthrin, sulfotep, tebupirimfos, temephos, terbufos, tetrachlorvinphos, thiometon, triazophos, alanycarb, aldicarb, bendiocarb, benfluracarb, butocarboxim, butoxycarboxim, carbaryl, carbofuran, carbosulfan, ethiofencarb, fenobucarb, formetanate, furathiocarb, isoprocarb, methiocarb, methymyl, metolcarb, oxamyl, primicarb, propoxur, thiodicarb, thiofanox, triazamate, trimethacarb, XMC, xylylcarb, acephate, azamethiphos, azinphos-ethyl, azinphos-methyl, cadusafos, chlorethoxyfox, trichlorfon, vamidothion, chlordane, endosulfan, ethiprole, flpronil, acrinathrin, allethrin, bifenthrin, bioallethrin, bioalletherin X-cyclopentenyl, bioresmethrin, cyclorothrin, cyfluthrin, cyhalothrin, cypermethrin, cyphenothrin [(1R)-trans-isomers], deltamethrin, empenthrin [(EZ)-(1R)-isomers], esfenvalerate, etofenprox, fenpropathrin, fenvalerate, flucythrinate, flumethrin, halfenprox, kadathrin, phenothrin [(1R)-trans-isomer] prallethrin, pyrethrins (pyrethrum), resmethrin, silafluofen, tefluthrin, tetramethrin, tetramethrin [(1R)-isomers], tralomethrin, transfluthrin, alpha-cypermethrin, beta-cyfluthrin, beta-cypermethrin, d-cis-trans allethrin, d-trans allethrin, gamma-cyhalothrin, lamda-cyhalothrin, tau-fluvalinate, theta-cypermethrin, zeta-cypermethrin, methoxychlor, nicotine, sulfoxaflor, acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid, thiamethoxan, tebuprimphos, beta-cyfluthrin, clothianidin, flonicamid, hydramethylnon, amitraz, flubendiamide, blorantraniliprole, lambda cyhalothrin, spinosad, gamma cyhalothrin, Beauveria bassiana, capsicum oleoresin extract, garlic oil, carbaryl, chlorpyrifos, sulfoxaflor, lambda cyhalothrin, Chlorfenvinphos, Chlormephos, Chlorpyrifos, Chlorpyrifos-methyl, Coumaphos, Cyanophos, Demeton-S-methyl, Diazinon, Dichlorvos/DDVP, Dicrotophos, Dimethoate, Dimethylvinphos, Disulfoton, EPN, Ethion, Ethoprophos, Famphur, Fenamiphos, Fenitrothion, Fenthion, Fosthiazate, Heptenophos, Imicyafos, Isofenphos, Isopropyl O-(methoxyaminothio-phosphoryl) salicylate, Isoxathion, Malathion, Mecarbam, Methamidophos, Methidathion, Mevinphos, Monocrotophos, Naled, Omethoate, Oxydemeton-methyl, Parathion, Parathion-methyl, Phenthoate, Phorate, Phosalone, Phosmet, Phosphamidon, Phoxim, Pirimiphos-methyl, Profenofos, Propetamphos, Prothiofos, Pyraclofos, Pyridaphenthion, Quinalphosfluacrypyrim, tebufenozide, chlorantraniliprole. Bacillus thuringiensis subs. Kurstaki, terbufos, mineral oil, fenpropathrin, metaldehyde, deltamethrin, diazinon, dimethoate, diflubenzuron, pyriproxyfen, reosemary oil, peppermint oil, geraniol, azadirachtin, piperonyl butoxide, cyantraniliprole, alpha cypermethrin, tefluthrin, pymetrozine, malathion, Bacillus thuringiensis subsp. israelensis, dicofol, bromopropylate, benzoximate, azadirachtin, flonicamid, soybean oil, Chromobacterium subtsugae strain PRAA4-1, zeta cypermethrin, phosmet, methoxyfenozide, paraffinic oil, spirotetramat, methomyl, Metarhizium anisopliae strain F52, ethoprop, tetradifon, propargite, fenbutatin oxide, azocyclotin, cyhexatin, diafenthiuron, Bacillus sphaericus, etoxazole, flupyradifurone, azadirachtin, Beauveria bassiana, cyflumetofen, azadirachtin, chinomethionat, acephate, Isaria fumosorosea Apopka strain 97, sodium tetraborohydrate decahydrate, emamectin benzoate, cryolite, spinetoram, Chenopodium ambrosioides extract, novaluron, dinotefuran, carbaryl, acequinocyl, flupyradifurone, iron phosphate, kaolin, buprofezin, cyromazine, chromafenozide, halofenozide, methoxyfenozide, tebufenozide, bistrifluron, chlorfluazuron, diflubenzuron, flucycloxuron, flufenoxuron, hexaflumuron, lufenuron, nocaluron, noviflumuron, teflubenzuron, tnflumuron, bensultap, cartap hydrochloride, thiocyclam, thiosultap-sodium, DNOC, chlorfenapyr, sulfuramid, phorate, tolfenpyrad, sulfoxaflor, neem oil, Bacillus thuringiensis subsp. tenebrionis strain SA-10, cyromazine, heat-killed Burkholderia spp., cyantraniliprole, cyenopyrafen, cyflumetofen, sodium cyanide, potassium cyanide, calcium cyanide, aluminum phosphide, calcium phosphide, phosphine, zinc phosphide, spriodiclofen, spiromesifen, spirotetramat, metaflumizone, flubendiamide, pyflubumide, oxamyl, Bacillus thuringiensis subsp. aizawai, etoxazole, and esfenvalerate.

TABLE 9 Exemplary insecticides associated with various modes of action, which can be combined with microbes of the disclosure Physiological Exemplary function(s) Mode of Action Compound class insecticides affected acetylcholinesterase carbamates Alanycarb, Aldicarb, Nerve and (AChE) inhibitors Bendiocarb, muscle Benfuracarb, Butocarboxim, Butoxycarboxim, Carbaryl, Carbofuran, Carbosulfan, Ethiofencarb, Fenobucarb, Formetanate, Furathiocarb, Isoprocarb, Methiocarb, Methomyl, Metolcarb, Oxamyl, Pirimicarb, Propoxur, Thiodicarb, Thiofanox, Triazamate, Trimethacarb, XMC, Xylylcarb acetylcholinesterase organophosphates Acephate, Nerve and (AChE) inhibitors Azamethiphos, muscle Azinphos-ethyl, Azinphos-methyl, Cadusafos, Chlorethoxyfos, Chlorfenvinphos, Chlormephos, Chlorpyrifos, Chlorpyrifos-methyl, Coumaphos, Cyanophos, Demeton- S-methyl, Diazinon, Dichlorvos/DDVP, Dicrotophos, Dimethoate, Dimethylvinphos, Disulfoton, EPN, Ethion, Ethoprophos, Famphur, Fenamiphos, Fenitrothion, Fenthion, Fosthiazate, Heptenophos, Imicyafos, Isofenphos, Isopropyl O- (methoxyaminothio- phosphoryl) salicylate, Isoxathion, Malathion, Mecarbam, Methamidophos, Methidathion, Mevinphos, Monocrotophos, Naled, Omethoate, Oxydemeton-methyl, Parathion, Parathion- methyl, Phenthoate, Phorate, Phosalone, Phosmet, Phosphamidon, Phoxim, Pirimiphos- methyl, Profenofos, Propetamphos, Prothiofos, Pyraclofos, Pyridaphenthion, Quinalphos, Sulfotep, Tebupirimfos, Temephos, Terbufos, Tetrachlorvinphos, Thiometon, Triazophos, Trichlorfon, Vamidothion GABA-gated cyclodiene Chlordane, Endosulfan Nerve and chloride channel organochlorines muscle blockers GABA-gated phenylpyrazoles Ethiprole, Fipronil Nerve and chloride channel (Fiproles) muscle blockers sodium channel pyrethroids, Acrinathrin, Allethrin, Nerve and modulators pyrethrins Bifenthrin, Bioallethrin, muscle Bioallethrin S- cyclopentenyl, Bioresmethrin, Cycloprothrin, Cyfluthrin, Cyhalothrin, Cypermethrin, Cyphenothrin [(1R)- trans- isomers], Deltamethrin, Empenthrin [(EZ)- (1R)- isomers], Esfenvalerate, Etofenprox, Fenpropathrin, Fenvalerate, Flucythrinate, Flumethrin, Halfenprox, Kadathrin, Phenothrin [(1R)-trans- isomer], Prallethrin, Pyrethrins (pyrethrum), Resmethrin, Silafluofen, Tefluthrin, Tetramethrin, Tetramethrin [(1R)- isomers], Tralomethrin, Transfluthrin, alpha- Cypermethrin, beta- Cyfluthrin, beta- Cypermethrin, d-cis- trans Allethrin, d-trans Allethrin, gamma- Cyhalothrin, lambda- Cvhalothrin, tau- Fluvalinate, theta- Cypermethrin, zeta- Cypermethrin sodium channel DDT, DDT, methoxychlor Nerve and modulators methoxychlor muscle nicotinic neonicotinoids Acetamiprid, Nerve and acetylcholine Clothianidin, muscle receptor (nAChR) Dinotefuran, competitive Imidacloprid, modulators Nitenpyram, Thiacloprid, Thiamethoxam nicotinic nicotine nicotine Nerve and acetylcholine muscle receptor (nAChR) competitive modulators nicotinic sulfoximines sulfoxaflor Nerve and acetylcholine muscle receptor (nAChR) competitive modulators nicotinic butenolides Flupyradifurone Nerve and acetylcholine muscle receptor (nAChR) competitive modulators nicotinic spinosyns Spinetoram, Spinosad Nerve and acetylcholine muscle receptor (nAChR) allosteric modulators Glutamate-gated avermectins, Abamectin, Emamectin Nerve and chloride channel milbemycins benzoate, Lepimectin, muscle (GluCl) allosteric Milbemectin modulators juvenile hormone juvenile hormone Hydroprene, Growth mimics analogues Kinoprene, Methoprene juvenile hormone Fenoxycarb Fenoxycarb Growth mimics juvenile hormone Pyriproxyfen Pyriproxyfen Growth mimics miscellaneous non- alkyl halides Methyl bromide and Unknown or specific (multi-site) other alkyl halides non-specific inhibitors miscellaneous non- Chloropicrin Chloropicrin Unknown or specific (multi-site) non-specific inhibitors miscellaneous non- fluorides Cryolite, sulfuryl Unknown or specific (multi-site) fluoride non-specific inhibitors miscellaneous non- borates Borax, Boric acid, Unknown or specific (multi-site) Disodium octaborate, non-specific inhibitors Sodium borate, Sodium metaborate miscellaneous non- tartar emetic tartar emetic Unknown or specific (multi-site) non-specific inhibitors miscellaneous non- methyl Dazomet, Metam Unknown or specific (multi-site) isothiocyanate non-specific inhibitors generators modulators of Pyridine Pymetrozine, Nerve and chordotonal organs azomethine Pyrifluquinazon muscle derivatives mite growth Clofentezine, Clofentezine, Growth inhibitors Diflovidazin, Diflovidazin, Hexythiazox Hexythiazox mite growth Etoxazole Etoxazole Growth inhibitors microbial Bacillus Bt var. aizawai, Bt var. Midgut disruptors of insect thuringiensis and israelensis, Bt var. midgut membranes the insecticidal kurstaki, Bt var. proteins they tenebrionensis produce microbial Bacillus Bacillus sphaericus Midgut disruptors of insect sphaericus midgut membranes inhibitors of Diafenthiuron Diafenthiuron Respiration mitochondrial ATP synthase inhibitors of organotin Azocyclotin, Respiration mitochondrial ATP miticides Cyhexatin, Fenbutatin synthase oxide inhibitors of Propargite Propargite Respiration mitochondrial ATP synthase inhibitors of Tetradifon Tetradifon Respiration mitochondrial ATP synthase uncouplers of Chlorfenapyr, Chlorfenapyr, DNOC, Respiration oxidative DNOC, Sulfuramid phosphorylation via Sulfuramid disruption of the proton gradient Nicotinic nereistoxin Bensultap, Cartap Nerve and acetylcholine analogues hydrochloride, muscle receptor (nAChR) Thiocyclam, channel blockers Thiosultap-sodium inhibitors of chitin benzoylureas Bistrifluron, Growth biosynthesis, type 0 Chlorfluazuron, Diflubenzuron, Flucycloxuron, Flufenoxuron, Hexaflumuron, Lufenuron, Novaluron, Noviflumuron, Teflubenzuron, Triflumuron inhibitors of chitin Buprofezin Buprofezin Growth biosynthesis, type 1 moulting disruptor, Cyromazine Cyromazine Growth Dipteran ecdysone receptor diacylhydrazines Chromafenozide, Growth agonists Halofenozide, Methoxyfenozide, Tebufenozide octopamine Amitraz Amitraz Nerve and receptor agonists muscle mitochondrial Hydramethylnon Hydramethylnon Respiration complex III electron transport inhibitors mitochondrial Acequinocyl Acequinocyl Respiration complex III electron transport inhibitors mitochondrial Fluacrypyrim Fluacrypyrim Respiration complex III electron transport inhibitors mitochondrial Bifenazate Bifenazate Respiration complex III electron transport inhibitors mitochondrial Meti acaricides Fenazaquin, Respiration complex I electron and insecticides Fenpyroximate, transport inhibitors Pyridaben, Pyrimidifen, Tebufenpyrad, Tolfenpyrad mitochondrial Rotenone Rotenone Respiration complex I electron transport inhibitors voltage-dependent oxadiazines Indoxacarb Nerve and sodium channel muscle blockers voltage-dependent semicarbazones Metaflumizone Nerve and sodium channel muscle blockers inhibitors of acetyl tetronic and Spirodiclofen, Growth CoA carboxylase tetramic acid Spiromesifen, derivatives Spirotetramat mitochondrial phosphides Aluminium phosphide, Respiration complex IV Calcium phosphide, electron transport Phosphine, Zinc inhibitors phosphide mitochondrial cyanides Calcium cyanide, Respiration complex IV Potassium cyanide, electron transport Sodium cyanide inhibitors mitochondrial beta-ketonitrile Cyenopyrafen, Respiration complex II electron derivatives Cyflumetofen transport inhibitors mitochondrial carboxanilides Pyflubumide Respiration complex II electron transport inhibitors ryanodine receptor diamides Chlorantraniliprole, Nerve and modulators Cyantraniliprole, muscle Flubendiamide Chordotonal organ Flonicamid Flonicamid Nerve and modulators - muscle undefined target site compounds of Azadirachtin Azadirachtin Unknown unknown or uncertain mode of action compounds of Benzoximate Benzoximate Unknown unknown or uncertain mode of action compounds of Bromopropylate Bromopropylate Unknown unknown or uncertain mode of action compounds of Chinomethionat Chinomethionat Unknown unknown or uncertain mode of action compounds of Dicofol Dicofol Unknown unknown or uncertain mode of action compounds of lime sulfur lime sulfur Unknown unknown or uncertain mode of action compounds of Pyridalyl Pyridalyl Unknown unknown or uncertain mode of action compounds of sulfur sulfur Unknown unknown or uncertain mode of action

TABLE 10 Exemplary list of pesticides, which can be combined with microbes of the disclosure Category Compounds INSECTICIDES arsenical insecticides calcium arsenate copper acetoarsenite copper arsenate lead arsenate potassium arsenite sodium arsenite botanical insecticides allicin anabasine azadirachtin carvacrol d-limonene matrine nicotine nornicotine oxymatrine pyrethrins cinerins cinerin I cinerin II jasmolin I jasmolin II pyrethrin I pyrethrin II quassia rhodojaponin-III rotenone ryania sabadilla sanguinarine triptolide carbamate insecticides bendiocarb carbaryl benzofuranyl methylcarbamate insecticides benfuracarb carbofuran carbosulfan decarbofuran furathiocarb dimethylcarbamate insecticides dimetan dimetilan hyquincarb isolan pirimicarb pyramat pyrolan oxime carbamate insecticides alanycarb aldicarb aldoxycarb butocarboxim butoxycarboxim methomyl nitrilacarb oxamyl tazimcarb thiocarboxime thiodicarb thiofanox phenyl methylcarbamate insecticides allyxycarb aminocarb bufencarb butacarb carbanolate cloethocarb CPMC dicresyl dimethacarb dioxacarb EMPC ethiofencarb fenethacarb fenobucarb isoprocarb methiocarb metolcarb mexacarbate promacyl promecarb propoxur trimethacarb XMC xylylcarb diamide insecticides broflanilide chlorantraniliprole cyantraniliprole cyclaniliprole cyhalodiamide flubendiamide tetraniliprole dinitrophenol insecticides dinex dinoprop dinosam DNOC fluorine insecticides barium hexafluorosilicate cryolite flursulamid sodium fluoride sodium hexafluorosilicate sulfluramid formamidine insecticides amitraz chlordimeform formetanate formparanate medimeform semiamitraz fumigant insecticides acrylonitrile carbon disulfide carbon tetrachloride carbonyl sulfide chloroform chloropicrin cyanogen para-dichlorobenzene 1,2-dichloropropane dithioether ethyl formate ethylene dibromide ethylene dichloride ethylene oxide hydrogen cyanide methyl bromide methyl iodide methylchloroform methylene chloride naphthalene phosphine sodium tetrathiocarbonate sulfuryl fluoride tetrachloroethane inorganic insecticides borax boric acid calcium polysulfide copper oleate diatomaceous earth mercurous chloride potassium thiocyanate silica gel sodium thiocyanate insect growth regulators chitin synthesis inhibitors buprofezin cyromazine benzoylphenylurea chitin synthesis bistrifluron inhibitors chlorbenzuron chlorfluazuron dichlorbenzuron diflubenzuron flucycloxuron flufenoxuron hexaflumuron lufenuron novaluron noviflumuron penfluron teflubenzuron triflumuron juvenile hormone mimics dayoutong epofenonane fenoxycarb hydroprene kinoprene methoprene pyriproxyfen triprene juvenile hormones juvenile hormone I juvenile hormone II juvenile hormone III moulting hormone agonists chromafenozide furan tebufenozide halofenozide methoxyfenozide tebufenozide yishijing moulting hormones α-ecdysone ecdysterone moulting inhibitors diofenolan precocenes precocene I precocene II precocene III unclassified insect growth regulators dicyclanil macrocyclic lactone insecticides avermectin insecticides abamectin doramectin emamectin eprinomectin ivermectin selamectin milbemycin insecticides lepimectin milbemectin milbemycin oxime moxidectin spinosyn insecticides spinetoram spinosad neonicotinoid insecticides nitroguanidine neonicotinoid insecticides clothianidin dinotefuran imidacloprid imidaclothiz thiamethoxam nitromethylene neonicotinoid insecticides nitenpyram nithiazine pyridylmethylamine neonicotinoid acetamiprid insecticides imidacloprid nitenpyram paichongding thiacloprid nereistoxin analogue insecticides bensultap cartap polythialan thiocyclam thiosultap organochlorine insecticides bromo-DDT camphechlor DDT pp′-DDT ethyl-DDD HCH gamma-HCH lindane methoxychlor pentachlorophenol TDE cyclodiene insecticides aldrin bromocyclen chlorbicyclen chlordane chlordecone dieldrin dilor endosulfan alpha-endosulfan endrin HEOD heptachlor HHDN isobenzan isodrin kelevan mirex organophosphorus insecticides organophosphate insecticides bromfenvinfos calvinphos chlorfenvinphos crotoxyphos dichlorvos dicrotophos dimethylvinphos fospirate heptenophos methocrotophos mevinphos monocrotophos naled naftalofos phosphamidon propaphos TEPP tetrachlorvinphos organothiophosphate insecticides dioxabenzofos fosmethilan phenthoate aliphatic organothiophosphate insecticides acethion acetophos amiton cadusafos chlorethoxyfos chlormephos demephion demephion-O demephion-S demeton demeton-O demeton-S demeton-methyl demeton-O-methyl demeton-S-methyl demeton-S-methylsulphon disulfoton ethion ethoprophos IPSP isothioate malathion methacrifos methylacetophos oxydemeton-methyl oxydeprofos oxydisulfoton phorate sulfotep terbufos thiometon aliphatic amide organothiophosphate amidithion insecticides cyanthoate dimethoate ethoate-methyl formothion mecarbam omethoate prothoate sophamide vamidothion oxime organothiophosphate insecticides chlorphoxim phoxim phoxim-methyl heterocyclic organothiophosphate azamethiphos insecticides colophonate coumaphos coumithoate dioxathion endothion menazon morphothion phosalone pyraclofos pyrazothion pyridaphenthion quinothion benzothiopyran organothiophosphate dithicrofos insecticides thicrofos benzotriazine organothiophosphate azinphos-ethyl insecticides azinphos-methyl isoindole organothiophosphate insecticides dialifos phosmet isoxazole organothiophosphate insecticides isoxathion zolaprofos pyrazolopyrimidine organothiophosphate chlorprazophos insecticides pyrazophos pyridine organothiophosphate insecticides chlorpyrifos chlorpyrifos-methyl pyrimidine organothiophosphate butathiofos insecticides diazinon etrimfos lirimfos pirimioxyphos pirimiphos-ethyl pirimiphos-methyl primidophos pyrimitate tebupirimfos quinoxaline organothiophosphate quinalphos insecticides quinalphos-methyl thiadiazole organothiophosphate athidathion insecticides lythidathion methidathion prothidathion triazole organothiophosphate insecticides isazofos triazophos phenyl organothinphosphate insecticides azothoate bromophos bromophos-ethyl carbophenothion chlorthiophos cyanophos cythioate dicapthon dichlofenthion etaphos famphur fenchlorphos fenitrothion fensulfothion fenthion fenthion-ethyl heterophos jodfenphos mesulfenfos parathion parathion-methyl phenkapton phosnichlor profenofos prothiofos sulprofos temephos trichlormetaphos-3 trifenofos xiaochongliulin phosphonate insecticides butonate trichlorfon phosphonothioate insecticides mecarphon phenyl ethylphosphonothioate insecticides fonofos trichloronat phenyl phenylphosphonothioate cyanofenphos insecticides EPN leptophos phosphoramidate insecticides crufomate fenamiphos fosthietan mephosfolan phosfolan phosfolan-methyl pirimetaphos phosphoramidothioate insecticides acephate chloramine phosphorus isocarbophos isofenphos isofenphos-methyl methamidophos phosglycin propetamphos phosphorodiamide insecticides dimefox mazidox mipafox schradan oxadiazine insecticides indoxacarb oxadiazolone insecticides metoxadiazone phthalimide insecticides dialifos phosmet tetramethrin physical insecticides maltodextrin desiccant insecticides boric acid diatomaceous earth silica gel pyrazole insecticides chlorantraniliprole cyantraniliprole cyclaniliprole dimetilan isolan tebufenpyrad tetraniliprole tolfenpyrad phenylpyrazole insecticides acetoprole ethiprole fipronil flufiprole pyraclofos pyrafluprole pyriprole pyrolan vaniliprole pyrethroid insecticides pyrethroid ester insecticides acrinathrin allethrin bioallethrin esdépalléthrine barthrin bifenthrin kappa-bifenthrin bioethanomethrin brofenvalerate brofluthrinate bromethrin butethrin chlorempenthrin cyclethrin cycloprothrin cyfluthrin beta-cyfluthrin cyhalothrin gamma-cyhalothrin lambda-cyhalothrin cypermethrin alpha-cypermethrin beta-cypermethrin theta-cypermethrin zeta-cypermethrin cyphenothrin deltamethrin dimefluthrin dimethrin empenthrin d-fanshiluquebingjuzhi chloroprallethrin fenfluthrin fenpirithrin fenpropathrin fenvalerate esfenvalerate flucythrinate fluvalinate tau-fluvalinate furamethrin furethrin heptafluthrin imiprothrin japothrins kadethrin methothrin metofluthrin epsilon-metofluthrin momfluorothrin epsilon-momfluorothrin pentmethrin permethrin biopermethrin transpermethrin phenothrin prallethrin profluthrin proparthrin pyresmethrin renofluthrin meperfluthrin resmethrin bioresmethrin cismethrin tefluthrin kappa-tefluthrin terallethrin tetramethrin tetramethylfluthrin tralocythrin tralomethrin transfluthrin valerate pyrethroid ether insecticides etofenprox flufenprox halfenprox protrifenbute silafluofen pyrethroid oxime insecticides sulfoxime thiofluoximate pyrimidinamine insecticides flufenerim pyrimidifen pyrrole insecticides chlorfenapyr quaternary ammonium insecticides sanguinarine sulfoximine insecticides sulfoxaflor tetramic acid insecticides spirotetramat tetronic acid insecticides spiromesifen thiazole insecticides clothianidin imidaclothiz thiamethoxam thiapronil thiazolidine insecticides tazimcarb thiacloprid thiourea insecticides diafenthiuron urea insecticides flucofuron sulcofuron zwitterionic insecticides dicloromezotiaz triflumezopyrim unclassified insecticides afidopyropen afoxolaner allosamidin closantel copper naphthenate crotamiton EXD fenazaflor fenoxacrim flometoquin flonicamid fluhexafon flupyradifurone fluralaner fluxametamide hydramethylnon isoprothiolane jiahuangchongzong malonoben metaflumizone nifluridide plifenate pyridaben pyridalyl pyrifluquinazon rafoxanide thuringiensin triarathene triazamate ACARICIDES botanical acaricides carvacrol sanguinarine bridged diphenyl acaricides azobenzene benzoximate benzyl benzoate bromopropylate chlorbenside chlorfenethol chlorfenson chlorfensulphide chlorobenzilate chloropropylate cyflumetofen DDT dicofol diphenyl sulfone dofenapyn fenson fentrifanil fluorbenside genit hexachlorophene phenproxide proclonol tetradifon tetrasul carbamate acaricides benomyl carbanolate carbaryl carbofuran methiocarb metolcarb promacyl propoxur oxime carbamate acaricides aldicarb butocarboxim oxamyl thiocarboxime thiofanox carbazate acaricides bifenazate dinitrophenol acaricides binapacryl dinex dinobuton dinocap dinocap-4 dinocap-6 dinocton dinopenton dinosulfon dinoterbon DNOC formamidine acaricides amitraz chlordimeform chloromebuform formetanate formparanate medimeform semiamitraz macrocyclic lactone acaricides tetranactin avermectin acaricides abamectin doramectin eprinomectin ivermectin selamectin milbemycin acaricides milbemectin milbemycin oxime moxidectin mite growth regulators clofentezine cyromazine diflovidazin dofenapyn fluazuron flubenzimine flucycloxuron flufenoxuron hexythiazox organochlorine acaricides bromocyclen camphechlor DDT dienochlor endosulfan lindane organophosphorus acaricides organophosphate acaricides chlorfenvinphos crotoxyphos dichlorvos heptenophos mevinphos monocrotophos naled TEPP tetrachlorvinphos organothiophosphate acaricides amidithion amiton azinphos-ethyl azinphos-methyl azothoate benoxafos bromophos bromophos-ethyl carbophenothion chlorpyrifos chlorthiophos coumaphos cyanthoate demeton demeton-O demeton-S demeton-methyl demeton-O-methyl demeton-S-methyl demeton-S-methylsulphon dialifos diazinon dimethoate dioxathion disulfoton endothion ethion ethoate-methyl formothion malathion mecarbam methacrifos omethoate oxydeprofos oxydisulfoton parathion phenkapton phorate phosalone phosmet phostin phoxim pirimiphos-methyl prothidathion prothoate pyrimitate quinalphos qumtiofos sophamide sulfotep thiometon triazophos trifenofos vamidothion phosphonate acaricides trichlorfon phosphoramidothioate acaricides isocarbophos methamidophos propetamphos phosphorodiamide acaricides dimefox mipafox schradan organotin acaricides azocyclotin cyhexatin fenbutatin oxide phostin phenylsulfamide acaricides dichlofluanid phthalimide acaricides dialifos phosmet pyrazole acaricides cyenopyrafen fenpyroximate pyflubumide tebufenpyrad phenylpyrazole acaricides acetoprole fipronil vaniliprole pyrethroid acaricides pyrethroid ester acaricides acrinathrin bifenthrin brofluthrinate cyhalothrin cypermethrin alpha-cypermethrin fenpropathrin fenvalerate flucythrinate flumethrin fluvalinate tau-fluvalinate permethrin pyrethroid ether acaricides halfenprox pyrimidinamine acaricides pyrimidifen pyrrole acaricides chlorfenapyr quaternary ammonium acaricides sanguinarine quinoxaline acaricides chinomethionat thioquinox strobilurin acaricides methoxyacrylate strobilurin acaricides bifujunzhi fluacrypyrim flufenoxystrobin pyriminostrobin sulfite ester acaricides aramite propargite tetronic acid acaricides spirodiclofen tetrazine acaricides clofentezine diflovidazin thiazolidine acaricides flubenzimine hexythiazox thiocarbamate acaricides fenothiocarb thiourea acaricides chloromethiuron diafenthiuron unclassified acaricides acequinocyl afoxolaner amidoflumet arsenous oxide clenpirin closantel crotamiton cycloprate cymiazole disulfiram etoxazole fenazaflor fenazaquin fluenetil fluralaner mesulfen MNAF nifluridide nikkomycins pyridaben sulfiram sulfluramid sulfur thuringiensin triarathene CHEMOSTERILANTS apholate bisazir busulfan diflubenzuron dimatif hemel hempa metepa methiotepa methyl apholate morzid penfluron tepa thiohempa thiotepa tretamine uredepa INSECT REPELLENTS acrep butopyronoxyl camphor d-camphor carboxide dibutyl phthalate diethyltoluamide dimethyl carbate dimethyl phthalate dibutyl succinate ethohexadiol hexamide icaridin methoquin-butyl methylneodecanamide 2-(octylthio)ethanol oxamate quwenzhi quyingding rebemide zengxiaoan NEMATICIDES avermectin nematicides abamectin botanical nematicides carvacrol carbamate nematicides benomyl carbofuran carbosulfan cloethocarb oxime carbamate nematicides alanycarb aldicarb aldoxycarb oxamyl tirpate fumigant nematicides carbon disulfide cyanogen 1,2-dichloropropane 1,3-dichloropropene dithioether methyl bromide methyl iodide sodium tetrathiocarbonate organophosphorus nematicides organophosphate nematicides diamidafos fenamiphos fosthietan phosphamidon organothiophosphate nematicides cadusafos chlorpyrifos dichlofenthion dimethoate ethoprophos fensulfothion fosthiazate heterophos isamidofos isazofos phorate phosphocarb terbufos thionazin triazophos phosphonothioate nematicides imicyafos mecarphon unclassified nematicides acetoprole benclothiaz chloropicrin dazomet DBCP DCIP fluazaindolizine fluensulfone furfural metam methyl isothiocyanate tioxazafen xylenols

Insecticides also include synergists or activators that are not in themselves considered toxic or insecticidal, but are materials used with insecticides to synergize or enhance the activity of the insecticides. Synergists or activators include piperonyl butoxide.

Biorational Pesticides

Insecticides can be biorational, or can also be known as biopesticides or biological pesticides. Biorational refers to any substance of natural origin (or man-made substances resembling those of natural origin) that has a detrimental or lethal effect on specific target pest(s), e.g., insects, weeds, plant diseases (including nematodes), and vertebrate pests, possess a unique mode of action, are non-toxic to man, domestic plants and animals, and have little or no adverse effects on wildlife and the environment.

Biorational insecticides (or biopesticides or biological pesticides) can be grouped as: (1) biochemicals (hormones, enzymes, pheromones and natural agents, such as insect and plant growth regulators), (2) microbial (viruses, bacteria, fungi, protozoa, and nematodes), or (3) Plant-Incorporated protectants (PIPs)—primarily transgenic plants, e.g., Bt corn.

Biopesticides, or biological pesticides, can broadly include agents manufactured from living microorganisms or a natural product and sold for the control of plant pests. Biopesticides can be: microorganisms, biochemicals, and semiochemicals. Biopesticides can also include peptides, proteins and nucleic acids such as double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA and hairpin DNA or RNA.

Bacteria, fungi, oomycetes, viruses and protozoa are all used for the biological control of insect pests. The most widely used microbial biopesticide is the insect pathogenic bacteria Bacillus thuringiensis (Bt), which produces a protein crystal (the Bt δ-endotoxin) during bacterial spore formation that is capable of causing lysis of gut cells when consumed by susceptible insects. Microbial Bt biopesticides consist of bacterial spores and δ-endotoxin crystals mass-produced in fermentation tanks and formulated as a sprayable product. Bt does not harm vertebrates and is safe to people, beneficial organisms and the environment. Thus, Bt sprays are a growing tactic for pest management on fruit and vegetable crops where their high level of selectivity and safety are considered desirable, and where resistance to synthetic chemical insecticides is a problem. Bt sprays have also been used on commodity crops such as maize, soybean and cotton, but with the advent of genetic modification of plants, farmers are increasingly growing Bt transgenic crop varieties.

Other microbial insecticides include products based on entomopathogenic baculoviruses. Baculoviruses that are pathogenic to arthropods belong to the virus family and possess large circular, covalently closed, and double-stranded DNA genomes that are packaged into nucleocapsids. More than 700 baculoviruses have been identified from insects of the orders Lepidoptera, Hymenoptera, and Diptera. Baculoviruses are usually highly specific to their host insects and thus, are safe to the environment, humans, other plants, and beneficial organisms. Over 50 baculovirus products have been used to control different insect pests worldwide. In the US and Europe, the Cydia pomonella granulovirus (CpGV) is used as an inundative biopesticide against codlingmoth on apples. Washington State, as the biggest apple producer in the US, uses CpGV on 13% of the apple crop. In Brazil, the nucleopolyhedrovirus of the soybean caterpillar Anticarsia gemmatalis was used on up to 4 million ha (approximately 35%) of the soybean crop in the mid-1990s. Viruses such as Gemstar® (Certis USA) are available to control larvae of Heliothis and Helicoverpa species.

At least 170 different biopesticide products based on entomopathogenic fungi have been developed for use against at least five insect and acarine orders in glasshouse crops, fruit and field vegetables as well as commodity crops. The majority of products are based on the ascomycetes Beauveria bassiana or Metarhizium anisopliae. M. anisopliae has also been developed for the control of locust and grasshopper pests in Africa and Australia and is recommended by the Food and Agriculture Organization of the United Nations (FAO) for locust management.

A number of microbial pesticides registered in the United States are listed in Table 16 of Kabaluk et al. 2010 (Kabaluk, J. T. et al. (ed). 2010. The Use and Regulation of Microbial Pesticides in Representative Jurisdictions Worldwide. IOBC Global. 99pp.) and microbial pesticides registered in selected countries are listed in Annex 4 of Hoeschle-Zeledon et al. 2013 (Hoeschle-Zeledon, I., P. Neuenschwander and L. Kumar. (2013). Regulatory Challenges for biological control. SP-IPM Secretariat International Institute of Tropical Agriculture (HTA), Ibadan, Nigeria. 43 pp.), each of which is incorporated herein in its entirety.

Plants produce a wide variety of secondary metabolites that deter herbivores from feeding on them. Some of these can be used as biopesticides. They include, for example, pyrethrins, which are fast-acting insecticidal compounds produced by Chrysanthemum cinerariaefolium. They have low mammalian toxicity but degrade rapidly after application. This short persistence prompted the development of synthetic pyrethrins (pyrethroids). The most widely used botanical compound is neem oil, an insecticidal chemical extracted from seeds of Azadirachta indica. Two highly active pesticides are available based on secondary metabolites synthesized by soil actinomycetes, but they have been evaluated by regulatory authorities as if they were synthetic chemical pesticides. Spinosad is a mixture of two macrolide compounds from Saccharopolyspora spinosa. It has a very low mammalian toxicity and residues degrade rapidly in the field. Farmers and growers used it widely following its introduction in 1997 but resistance has already developed in some important pests such as western flower thrips. Abamectin is a macrocyclic lactone compound produced by Streptomyces avermitilis. It is active against a range of pest species but resistance has developed to it also, for example, in tetranychid mites.

Peptides and proteins from a number of organisms have been found to possess pesticidal properties. Perhaps most prominent are peptides from spider venom (King, G. F. and Hardy, M. C. (2013) Spider-venom peptides: structure, pharmacology, and potential for control of insect pests. Annu. Rev. Entomol. 58: 475-496). A unique arrangement of disulfide bonds in spider venom peptides render them extremely resistant to proteases. As a result, these peptides are highly stable in the insect gut and hemolymph and many of them are orally active. The peptides target a wide range of receptors and ion channels in the insect nervous system. Other examples of insecticidal peptides include: sea anemone venom that act on voltage-gated Na+ channels (Bosnians, F. and Tytgat, J. (2007) Sea anemone venom as a source of insecticidal peptides acting on voltage-gated Na+ channels. Toxicon. 49(4): 550-560); the PA1b (Pea Albumin 1, subunit b) peptide from Legume seeds with lethal activity on several insect pests, such as mosquitoes, some aphids and cereal weevils (Eyraud, V. et al. (2013) Expression and Biological Activity of the Cystine Knot Bioinsecticide PA1b (Pea Albumin 1 Subunit b). PLoS ONE 8(12): e81619); and an internal 10 kDa peptide generated by enzymatic hydrolysis of Canavalia ensiformis (jack bean) urease within susceptible insects (Martinelli, A. H. S., et al. (2014) Structure-function studies on jaburetox, a recombinant insecticidal peptide derived from jack bean (Canavalia ensiformis) urease. Biochimica et Biophysica Acta 1840: 935-944). Examples of commercially available peptide insecticides include Spear™-T for the treatment of thrips in vegetables and ornamentals in greenhouses, Spear™-P to control the Colorado Potato Beetle, and Spear™-C to protect crops from lepidopteran pests (Vestaron Corporation, Kalamazoo, Mich.). A novel insecticidal protein from Bacillus bombysepticus, called parasporal crystal toxin (PC), shows oral pathogenic activity and lethality towards silkworms and Cry1Ac-resistant Helicoverpa armigera strains (Lin, P. et al. (2015) PC, a novel oral insecticidal toxin from Bacillus bombysepticus involved in host lethality via APN and BtR-175. Sci. Rep. 5: 11101).

A semiochemical is a chemical signal produced by one organism that causes a behavioral change in an individual of the same or a different species. The most widely used semiochemicals for crop protection are insect sex pheromones, some of which can now be synthesized and are used for monitoring or pest control by mass trapping, lure-and-kill systems and mating disruption. Worldwide, mating disruption is used on over 660,000 ha and has been particularly useful in orchard crops.

As used herein, “transgenic insecticidal trait” refers to a trait exhibited by a plant that has been genetically engineered to express a nucleic acid or polypeptide that is detrimental to one or more pests. In one embodiment, the plants of the present disclosure are resistant to attach and/or infestation from any one or more of the pests of the present disclosure. In one embodiment, the trait comprises the expression of vegetative insecticidal proteins (VIPs) from Bacillus thuringiensis, lectins and proteinase inhibitors from plants, terpenoids, cholesterol oxidases from Streptomyces spp., insect chitinases and fungal chitinolytic enzymes, bacterial insecticidal proteins and early recognition resistance genes. In another embodiment, the trait comprises the expression of a Bacillus thuringiensis protein that is toxic to a pest. In one embodiment the Bt protein is a Cry protein (crystal protein). Bt crops include Bt corn, Bt cotton and Bt soy. Bt toxins can be from the Cry family (see, for example, Crickmore et al., 1998, Microbiol. Mol. Biol. Rev. 62: 807-812), which are particularly effective against Lepidoptera, Coleoptera and Diptera.

Bt Cry and Cyt toxins belong to a class of bacterial toxins known as pore-forming toxins (PFT) that are secreted as water-soluble proteins undergoing conformational changes in order to insert into, or to translocate across, cell membranes of their host. There are two main groups of PFT: (i) the α-helical toxins, in which α-helix regions form the trans-membrane pore, and (ii) the β-barrel toxins, that insert into the membrane by forming a β-barrel composed of βsheet hairpins from each monomer. See, Parker M W, Feil S C, “Pore-forming protein toxins: from structure to function,” Prog. Biophys. Mol. Biol. 2005 May; 88(1):91-142. The first class of PFT includes toxins such as the colicins, exotoxin A, diphtheria toxin and also the Cry three-domain toxins. On the other hand, aerolysin, α-hemolysin, anthrax protective antigen, cholesterol-dependent toxins as the perfiringolysin O and the Cyt toxins belong to the β-barrel toxins. Id. In general, PFT producing-bacteria secrete their toxins and these toxins interact with specific receptors located on the host cell surface. In most cases, PFT are activated by host proteases after receptor binding inducing the formation of an oligomeric structure that is insertion competent Finally, membrane insertion is triggered, in most cases, by a decrease in pH that induces a molten globule state of the protein. Id.

The development of transgenic crops that produce Bt Cry proteins has allowed the substitution of chemical insecticides by environmentally friendly alternatives. In transgenic plants the Cry toxin is produced continuously, protecting the toxin from degradation and making it reachable to chewing and boring insects. Cry protein production in plants has been improved by engineering cry genes with a plant biased codon usage, by removal of putative splicing signal sequences and deletion of the carboxy-terminal region of the protoxin. See, Schuler T H, et al., “Insect-resistant transgenic plants,” Trends Biotechnol. 1998; 16:168-175. The use of insect resistant crops has diminished considerably the use of chemical pesticides in areas where these transgenic crops are planted. See, Qaim M, Zilberman D, “Yield effects of genetically modified crops in developing countries,” Science. 2003 Feb. 7; 299(5608):900-2.

Known Cry proteins include: δ-endotoxins including but not limited to: the Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry 28, Cry 29, Cry 30, Cry31, Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry 46, Cry47, Cry49, Cry 51, Cry52, Cry 53, Cry 54, Cry55, Cry56, Cry57, Cry58, Cry59. Cry60, Cry61, Cry62, Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70 and Cry71 classes of δ-endotoxin genes and the B. thuringiensis cytolytic cyt1 and cyt2 genes.

Members of these classes of B. thuringiensis insecticidal proteins include, but are not limited to: Cry1Aa1 (Accession #AAA22353); Cry1Aa2 (Accession #Accession #AAA22552); Cry1Aa3 (Accession #BAA00257); Cry1Aa4 (Accession #CAA31886); Cry1Aa5 (Accession #BAA04468); Cry1Aa6 (Accession #AAA86265); Cry1Aa7 (Accession #AAD46139); Cry1Aa8 (Accession #126149); Cry1Aa9 (Accession #BAA77213); Cry1Aa10 (Accession #AAD55382); Cry1Aa11 (Accession #CAA70856); Cry1Aa12 (Accession #AAP80146); Cry1Aa13 (Accession #AAM44305); Cry1Aa14 (Accession #AAP40639); Cry1Aa15 (Accession #AAY66993); Cry1Aa16 (Accession #HQ439776); Cry1Aa17 (Accession #HQ439788); Cry1Aa18 (Accession #HQ439790); Cry1Aa19 (Accession #HQ685121); Cry1Aa20 (Accession #JF340156); Cry1Aa21 (Accession #JN6514%); Cry1Aa22 (Accession #KC158223); Cry1Ab1 (Accession #AAA22330); Cry1Ab2 (Accession #AAA22613); Cry1Ab3 (Accession #AAA22561); Cry1Ab4 (Accession #BAA00071); Cry1Ab5 (Accession #CAA28405); Cry1Ab6 (Accession #AAA22420); Cry1Ab7 (Accession #CAA31620); Cry1Ab8 (Accession #AAA22551); Cry1Ab9 (Accession #CAA38701); Cry1Ab10 (Accession #A29125); Cry1Ab11 (Accession #112419); Cry1Ab12 (Accession #AAC64003); Cry1Ab13 (Accession #AAN76494); Cry1Ab14 (Accession #AAG16877); Cry1Ab15 (Accession #AA013302); Cry1Ab16 (Accession #AAK55546); Cry1Ab17 (Accession #AAT46415); Cry1Ab18 (Accession #AAQ88259); Cry1Ab19 (Accession #AAW31761), Cry1Ab20 (Accession #ABB72460); Cry1Ab21 (Accession #ABS18384), Cry1Ab22 (Accession #ABW87320), Cry1Ab23 (Accession #HQ439777); Cry1Ab24 (Accession #HQ439778); Cry1Ab25 (Accession #HQ685122); Cry1Ab26 (Accession #HQ847729), Cry1Ab27 (Accession #JN135249); Cry1Ab28 (Accession #JN135250); Cry1Ab29 (Accession #JN135251), Cry1Ab30 (Accession #JN135252); Cry1Ab31 (Accession #JN135253); Cry1Ab32 (Accession #JN135254), Cry1Ab33 (Accession #AAS93798); Cry1Ab34 (Accession #KC156668); Cry1Ab-like (Accession #AAK14336); Cry1Ab-like (Accession #AAK14337); Cry1Ab-like (Accession #AAK14338); Cry1Ab-like (Accession #ABG88858); Cry1Ac1 (Accession #AAA22331); Cry1Ac2 (Accession #AAA22338); Cry1Ac3 (Accession #CAA38098); Cry1Ac4 (Accession #AAA73077); Cry1Ac5 (Accession #AAA22339); Cry1Ac6 (Accession #AAA86266); Cry1Ac7 (Accession #AAB46989), Cry1Ac8 (Accession #AAC44841), Cry1Ac9 (Accession #AAB49768); Cry1Ac10 (Accession #CAA05505); Cry1Ac11 (Accession #CAA10270); Cry1Ac12 (Accession #112418); Cry1Ac13 (Accession #AAD38701); Cry1Ac14 (Accession #AAQ06607); Cry1Ac15 (Accession #AAN07788); Cry1Ac16 (Accession #AAU87037); Cry1Ac17 (Accession #AAX18704); Cry1Ac18 (Accession #AAY88347); Cry1Ac19 (Accession #ABD37053); Cry1Ac20 (Accession #ABB89046); Cry1Ac21 (Accession #AAY66992); Cry1Ac22 (Accession #ABZ01836); Cry1Ac23 (Accession #CAQ30431); Cry1Ac24 (Accession #ABL01535); Cry1Ac25 (Accession #FJ513324); Cry1Ac26 (Accession #FJ617446); Cry1Ac27 (Accession #FJ617447); Cry1Ac28 (Accession #ACM90319); Cry1Ac29 (Accession #DQ438941); Cry1Ac30 (Accession #GQ227507); Cry1Ac31 (Accession #GU446674); Cry1Ac32 (Accession #HM061081); Cry1Ac33 (Accession #GQ866913); Cry1Ac34 (Accession #HQ230364); Cry1Ac35 (Accession #JF340157); Cry1Ac36 (Accession #JN387137); Cry1Ac37 (Accession #JQ317685); Cry1Ad1 (Accession #AAA22340); Cry1Ad2 (Accession #CAA01880); Cry1Ae1 (Accession #AAA22410); Cry1Af1 (Accession #AAB82749); Cry1Ag1 (Accession #AAD46137); Cry1Ah1 (Accession #AAQ14326); Cry1AH2 (Accession #ABB76664); Cry1AH3 (Accession #HQ439779); Cry1Ai1 (Accession #AA039719); Cry1Ai2 (Accession #HQ439780); Cry1A-like (Accession #AAK14339); Cry1Ba1 (Accession #CAA29898); Cry1Ba2 (Accession #CAA65003); Cry1Ba3 (Accession #AAK63251); Cry1Ba4 (Accession #AAK51084); Cry1Ba5 (Accession #AB020894); Cry1Ba6 (Accession #ABL60921); Cry1Ba7 (Accession #HQ439781); Cry1Bb1 (Accession #AAA22344); Cry1Bb2 (Accession #HQ439782), Cry1Bc1 (Accession #CAA86568); Cry1Bd1 (Accession #AAD10292); Cry1Bd2 (Accession #AAM934%); Cry1Be1 (Accession #AAC32850), Cry1Be2 (Accession #AAQ52387), Cry1Be3 (Accession #ACV96720); Cry1Be4 (Accession #HM070026); Cry1Bf1 (Accession #CAC50778); Cry1Bf2 (Accession #AAQ52380); Cry1Bg1 (Accession #AA039720); Cry1Bh1 (Accession #HQ589331); Cry1Bi1 (Accession #KC156700); Cry1Ca1 (Accession #CAA30396); Cry1Ca2 (Accession #CAA31951); Cry1Ca3 (Accession #AAA22343); Cry1Ca4 (Accession #CAA01886), Cry1Ca5 (Accession #CAA65457); Cry1Ca6 [1] (Accession #AAF37224); Cry1Ca7 (Accession #AAG50438); Cry1Ca8 (Accession #AAM00264), Cry1Ca9 (Accession #AAL79362); Cry1Ca10 (Accession #AAN16462); Cry1Ca11 (Accession #AAX53094); Cry1Ca12 (Accession #HM070027); Cry1Ca13 (Accession #HQ412621), Cry1Ca14 (Accession #JN651493), Cry1Cb1 (Accession #M97880); Cry1Cb2 (Accession #AAG35409); Cry1Cb3 (Accession #ACD50894); Cry1Cb-like (Accession #AAX63901); Cry1Da1 (Accession #CAA38099); Cry1Da2 (Accession #176415); Cry1Da3 (Accession #HQ439784); Cry1 db1 (Accession #CAA80234); Cry1 db2 (Accession #AAK48937); Cry1 Del (Accession #ABK35074); Cry1Ea1 (Accession #CAA37933); Cry1Ea2 (Accession #CAA39609); Cry1Ea3 (Accession #AAA22345); Cry1Ea4 (Accession #AAD04732); Cry1Ea5 (Accession #A15535); Cry1Ea6 (Accession #AAL50330); Cry1Ea7 (Accession #AAW72936); Cry1Ea8 (Accession #ABX11258); Cry1Ea9 (Accession #HQ439785); Cry1Ea10 (Accession #ADR00398); Cry1Ea11 (Accession #JQ652456); Cry1Eb1 (Accession #AAA22346); Cry1Fa1 (Accession #AAA22348); Cry1Fa2 (Accession #AAA22347); Cry1Fa3 (Accession #HM070028); Cry1Fa4 (Accession #HM43%38); Cry1Fb1 (Accession #CAA80235); Cry1Fb2 (Accession #BAA25298); Cry1Fb3 (Accession #AAF21767); Cry1Fb4 (Accession #AAC10641); Cry1Fb5 (Accession #AA013295); Cry1Fb6 (Accession #ACD50892); Cry1Fb7 (Accession #ACD50893); Cry1Ga1 (Accession #CAA80233); Cry1Ga2 (Accession #CAA70506); Cry1Gb1 (Accession #AAD10291); Cry1Gb2 (Accession #AA013756); Cry1Gc1 (Accession #AAQ52381); Cry1Ha1 (Accession #CAA80236); Cry1Hb1 (Accession #AAA79694); Cry1Hb2 (Accession #HQ439786); Cry1H-like (Accession #AAF01213); Cry1Ia1 (Accession #CAA44633); Cry1Ia2 (Accession #AAA22354); Cry1Ia3 (Accession #AAC36999); Cry1Ia4 (Accession #AAB00958); Cry1Ia5 (Accession #CAA70124); Cry1Ia6 (Accession #AAC26910); Cry1Ia7 (Accession #AAM73516); Cry1Ia8 (Accession #AAK66742); Cry1Ia9 (Accession #AAQ08616); Cry1Ia10 (Accession #AAP86782), Cry1Ia11 (Accession #CAC85964), Cry1Ia12 (Accession #AAV53390); Cry1Ia13 (Accession #ABF83202); Cry1Ia14 (Accession #ACG63871); Cry1Ia15 (Accession #FJ617445); Cry1Ia16 (Accession #FJ617448); Cry1Ia17 (Accession #GU989199); Cry1Ia18 (Accession #ADK23801); Cry1Ia19 (Accession #HQ439787), Cry1Ia20 (Accession #JQ228426); Cry1Ia21 (Accession #JQ228424), Cry1Ia22 (Accession #JQ228427); Cry1Ia23 (Accession #JQ228428); Cry1Ia24 (Accession #JQ228429); Cry1Ia25 (Accession #JQ228430); Cry1Ia26 (Accession #JQ228431); Cry1Ia27 (Accession #JQ228432); Cry1Ia28 (Accession #JQ228433); Cry1Ia29 (Accession #JQ228434); Cry1Ia30 (Accession #JQ317686); Cry1Ia31 (Accession #JX944038); Cry1Ia32 (Accession #JX944039); Cry1Ia33 (Accession #JX944040), Cry1Ib1 (Accession #AAA82114); Cry1Ib2 (Accession #ABW88019); Cry1Ib3 (Accession #ACD75515); Cry1Ib4 (Accession #HM051227), Cry1Ib5 (Accession #HM070028); Cry1Ib6 (Accession #ADK38579); Cry1Ib7 (Accession #JN571740); Cry1Ib8 (Accession #JN675714); Cry1Ib9 (Accession #JN675715); Cry1Ib10 (Accession #JN675716); Cry1Ib11 (Accession #JQ228423); Cry1Ic1 (Accession #AAC62933); Cry1Ic2 (Accession #AAE71691); Cry1Id1 (Accession #AAD44366); Cry1Id2 (Accession #JQ228422); Cry1Ie1 (Accession #AAG43526); Cry1Ie2 (Accession #HM439636); Cry1Ie3 (Accession #KC156647); Cry1Ie4 (Accession #KC156681); Cry1If1 (Accession #AAQ52382); Cry1Ig1 (Accession #KC156701); Cry1I-like (Accession #AAC31094); Cry1I-like (Accession #ABG88859); Cry1Ja1 (Accession #AAA22341); Cry1 Ja2 (Accession #HM070030); Cry1Ja3 (Accession #JQ228425); Cry1Jb1 (Accession #AAA98959); Cry1Jc1 (Accession #AAC31092); Cry1Jc2 (Accession #AAQ52372); Cry1Jd1 (Accession #CAC50779); Cry1Ka1 (Accession #AAB00376); Cry1Ka2 (Accession #HQ439783); Cry1La1 (Accession #AAS60191); Cry1La2 (Accession #HM070031); Cry1Ma1 (Accession #FJ884067); Cry1Ma2 (Accession #KC156659); Cry1Na1 (Accession #KC156648); Cry1Nb1 (Accession #KC156678); Cry1-like (Accession #AAC31091); Cry2Aa1 (Accession #AAA22335); Cry2Aa2 (Accession #AAA83516); Cry2Aa3 (Accession #D86064); Cry2Aa4 (Accession #AAC04867); Cry2Aa5 (Accession #CAA10671); Cry2Aa6 (Accession #CAA10672); Cry2Aa7 (Accession #CAA10670); Cry2Aa8 (Accession #AA013734); Cry2Aa9 (Accession #AA013750); Cry2Aa10 (Accession #AAQ04263); Cry2Aa11 (Accession #AAQ52384); Cry2Aa12 (Accession #AB183671); Cry2Aa13 (Accession #ABL01536); Cry2Aa14 (Accession #ACF04939); Cry2Aa15 (Accession #JN426947); Cry2Ab1 (Accession #AAA22342), Cry2Ab2 (Accession #CAA39075), Cry2Ab3 (Accession #AAG36762); Cry2Ab4 (Accession #AA013296), Cry2Ab5 (Accession #AAQ04609); Cry2Ab6 (Accession #AAP59457); Cry2Ab7 (Accession #AAZ66347); Cry2Ab8 (Accession #ABC95996), Cry2Ab9 (Accession #ABC74968); Cry2Ab10 (Accession #EF157306); Cry2Ab11 (Accession #CAM84575); Cry2Ab12 (Accession #ABM21764), Cry2Ab13 (Accession #ACG76120), Cry2Ab14 (Accession #ACG76121); Cry2Ab15 (Accession #HM037126); Cry2Ab16 (Accession #GQ866914); Cry2Ab17 (Accession #HQ439789); Cry2Ab18 (Accession #JN135255); Cry2Ab19 (Accession #JN135256); Cry2Ab20 (Accession #JN135257); Cry2Ab21 (Accession #JN135258); Cry2Ab22 (Accession #JN135259), Cry2Ab23 (Accession #JN135260); Cry2Ab24 (Accession #JN135261); Cry2Ab25 (Accession #JN415485); Cry2Ab26 (Accession #JN426946); Cry2Ab27 (Accession #JN415764); Cry2Ab28 (Accession #JN651494); Cry2Ac1 (Accession #CAA40536); Cry2Ac2 (Accession #AAG35410); Cry2Ac3 (Accession #AAQ52385); Cry2Ac4 (Accession #ABC95997); Cry2Ac5 (Accession #ABC74969); Cry2Ac6 (Accession #ABC74793); Cry2Ac7 (Accession #CAL18690); Cry2Ac8 (Accession #CAM09325); Cry2Ac9 (Accession #CAM09326); Cry2Ac10 (Accession #ABN15104); Cry2Ac11 (Accession #CAM83895); Cry2Ac12 (Accession #CAM83896); Cry2Ad1 (Accession #AAF09583); Cry2Ad2 (Accession #ABC86927); Cry2Ad3 (Accession #CAK29504); Cry2Ad4 (Accession #CAM32331); Cry2Ad5 (Accession #CA078739); Cry2Ae1 (Accession #AAQ52362); Cry2Af1 (Accession #AB030519); Cry2Af2 (Accession #GQ866915); Cry2Ag1 (Accession #ACH91610); Cry2Ah1 (Accession #EU939453); Cry2Ah2 (Accession #ACL80665); Cry2Ah3 (Accession #GU073380); Cry2Ah4 (Accession #KC156702); Cry2Ai1 (Accession #FJ788388); Cry2Aj (Accession #); Cry2Ak1 (Accession #KC156660); Cry2Ba1 (Accession #KC156658); Cry3Aa1 (Accession #AAA22336); Cry3Aa2 (Accession #AAA22541); Cry3Aa3 (Accession #CAA68482); Cry3Aa4 (Accession #AAA22542); Cry3Aa5 (Accession #AAA50255); Cry3Aa6 (Accession #AAC43266); Cry3Aa7 (Accession #CAB41411); Cry3Aa8 (Accession #AAS79487); Cry3Aa9 (Accession #AAW05659); Cry3Aa10 (Accession #AAU29411); Cry3Aa11 (Accession #AAW82872); Cry3Aa12 (Accession #ABY49136); Cry3Ba1 (Accession #CAA34983); Cry3Ba2 (Accession #CAA00645); Cry3Ba3 (Accession #JQ397327); Cry3Bb1 (Accession #AAA22334); Cry3Bb2 (Accession #AAA74198); Cry3Bb3 (Accession #115475); Cry3Ca1 (Accession #CAA42469); Cry4Aa1 (Accession #CAA68485); Cry4Aa2 (Accession #BAA00179); Cry4Aa3 (Accession #CAD30148), Cry4Aa4 (Accession #AFB18317); Cry4A-like (Accession #AAY96321); Cry4Ba1 (Accession #CAA30312); Cry4Ba2 (Accession #CAA30114), Cry4Ba3 (Accession #AAA22337); Cry4Ba4 (Accession #BAA00178), Cry4Ba5 (Accession #CAD30095); Cry4Ba-like (Accession #ABC47686); Cry4Ca1 (Accession #EU646202); Cry4Cb1 (Accession #FJ403208), Cry4Cb2 (Accession #FJ597622); Cry4Cc1 (Accession #FJ403207), Cry5Aa1 (Accession #AAA67694); Cry5Ab1 (Accession #AAA67693), Cry5Ac1 (Accession #134543); Cry5Ad1 (Accession #ABQ82087); Cry5Ba1 (Accession #AAA68598); Cry5Ba2 (Accession #ABW88931); Cry5Ba3 (Accession #AFJ04417); Cry5Ca1 (Accession #HM461869); Cry5Ca2 (Accession #ZP_04123426); Cry5Da1 (Accession #HM461870); Cry5Da2 (Accession #ZP_04123980); Cry5Ea1 (Accession #HM485580); Cry5Ea2 (Accession #ZP_04124038); Cry6Aa1 (Accession #AAA22357); Cry6Aa2 (Accession #AAM46849); Cry6Aa3 (Accession #ABH03377); Cry6Ba1 (Accession #AAA22358); Cry7Aa1 (Accession #AAA22351); Cry7Ab1 (Accession #AAA21120); Cry7Ab2 (Accession #AAA21121); Cry7Ab3 (Accession #ABX24522); Cry7 Ab4 (Accession #EU380678); Cry7 Ab5 (Accession #ABX79555); Cry7 Ab6 (Accession #ACI44005); Cry7 Ab7 (Accession #ADB89216); Cry7 Ab8 (Accession #GUI45299); Cry7Ab9 (Accession #ADD92572); Cry7Ba1 (Accession #ABB70817); Cry7Bb1 (Accession #KC156653); Cry7Ca1 (Accession #ABR67863); Cry7Cb1 (Accession #KC156698); Cry7Da1 (Accession #ACQ99547); Cry7Da2 (Accession #HM572236); Cry7Da3 (Accession #KC156679); Cry7Ea1 (Accession #HM035086); Cry7Ea2 (Accession #HM132124); Cry7Ea3 (Accession #EEM19403); Cry7Fa1 (Accession #HM035088); Cry7Fa2 (Accession #EEM19090); Cry7Fb1 (Accession #HM572235); Cry7Fb2 (Accession #KC156682); Cry7Ga1 (Accession #HM572237); Cry7Ga2 (Accession #KC156669); Cry7Gb1 (Accession #KC156650); Cry7Gc1 (Accession #KC156654); Cry7Gd1 (Accession #KC156697); Cry7Ha1 (Accession #KC156651); Cry7Ia1 (Accession #KC156665); Cry7Ja1 (Accession #KC156671); Cry7Ka1 (Accession #KC156680); Cry7Kb1 (Accession #BAM99306); Cry7La1 (Accession #BAM99307); Cry8Aa1 (Accession #AAA21117); Cry8Ab1 (Accession #EU044830); Cry8Ac1 (Accession #KC156662); Cry8Ad1 (Accession #KC156684); Cry8Ba1 (Accession #AAA21118); Cry8Bb1 (Accession #CAD57542); Cry8Bc1 (Accession #CAD57543); Cry8Ca1 (Accession #AAA21119); Cry8Ca2 (Accession #AAR98783); Cry8Ca3 (Accession #EU625349); Cry8Ca4 (Accession #ADB54826); Cry8Da1 (Accession #BAC07226); Cry8Da2 (Accession #BD133574); Cry8Da3 (Accession #BD133575), Cry8db1 (Accession #BAF93483); Cry8Ea1 (Accession #AAQ73470), Cry8Ea2 (Accession #EU047597); Cry8Ea3 (Accession #KC855216); Cry8Fa1 (Accession #AAT48690); Cry8Fa2 (Accession #HQ174208); Cry8Fa3 (Accession #AFH78109), Cry8Ga1 (Accession #AAT46073); Cry8Ga2 (Accession #ABC42043); Cry8Ga3 (Accession #FJ198072); Cry8Ha1 (Accession #AAW81032), Cry8Ia1 (Accession #EU381044); Cry8Ia2 (Accession #GU073381); Cry8Ia3 (Accession #HM044664); Cry8Ia4 (Accession #KC156674); Cry8Ib1 (Accession #GU325772); Cry8Ib2 (Accession #KC156677); Cry8Ja1 (Accession #EU625348), Cry8Ka1 (Accession #FJ422558); Cry8Ka2 (Accession #ACN87262); Cry8Kb1 (Accession #HM123758); Cry8Kb2 (Accession #KC156675); Cry8La1 (Accession #GU325771); Cry8Ma1 (Accession #HM044665); Cry8Ma2 (Accession #EEM86551); Cry8Ma3 (Accession #HM210574); Cry8Na1 (Accession #HM640939); Cry8Pa1 (Accession #HQ388415); Cry8Qa1 (Accession #HQ441166); Cry8Qa2 (Accession #KC152468); Cry8Ra1 (Accession #AFP87548); Cry8Sa1 (Accession #JQ740599); Cry8Ta1 (Accession #KC156673); Cry8-like (Accession #FJ770571); Cry8-like (Accession #ABS53003); Cry9Aa1 (Accession #CAA41122); Cry9Aa2 (Accession #CAA41425); Cry9Aa3 (Accession #GQ249293); Cry9Aa4 (Accession #GQ249294); Cry9Aa5 (Accession #JX174110); Cry9Aa like (Accession #AAQ52376); Cry9Ba1 (Accession #CAA52927); Cry9Ba2 (Accession #GU299522); Cry9Bb1 (Accession #AAV28716); Cry9Ca1 (Accession #CAA85764); Cry9Ca2 (Accession #AAQ52375); Cry9Da1 (Accession #BAA19948); Cry9Da2 (Accession #AAB97923); Cry9Da3 (Accession #GQ249293); Cry9Da4 (Accession #GQ249297); Cry9db1 (Accession #AAX78439); Cry9Dc1 (Accession #KCI56683); Cry9Ea1 (Accession #BAA34908); Cry9Ea2 (Accession #AA012908); Cry9Ea3 (Accession #ABM21765); Cry9Ea4 (Accession #ACE88267); Cry9Ea5 (Accession #ACF04743); Cry9Ea6 (Accession #ACG63872); Cry9Ea7 (Accession #FJ380927); Cry9Ea8 (Accession #GQ249292); Cry9Ea9 (Accession #JN651495); Cry9Eb1 (Accession #CAC50780); Cry9Eb2 (Accession #GQ249298); Cry9Eb3 (Accession #KC156646); Cry9Ec1 (Accession #AAC63366); Cry9Ed1 (Accession #AAX78440); Cry9Ee1 (Accession #GQ249296); Cry9Ee2 (Accession #KC156664); Cry9Fa1 (Accession #KC156692); Cry9Ga1 (Accession #KC156699); Cry9-like (Accession #AAC63366); Cry10Aa1 (Accession #AAA22614); Cry10Aa2 (Accession #E00614); Cry10Aa3 (Accession #CAD30098); Cry10Aa4 (Accession #AFB18318); Cry10A-like (Accession #DQ167578), Cry11Aa1 (Accession #AAA22352); Cry11Aa2 (Accession #AAA22611), Cry11Aa3 (Accession #CAD30081); Cry11Aa4 (Accession #AFB18319); Cry11Aa-like (Accession #DQ166531); Cry11Ba1 (Accession #CAA60504); Cry11Bb1 (Accession #AAC97162); Cry11Bb2 (Accession #HM068615); Cry12Aa1 (Accession #AAA22355); Cry13Aa1 (Accession #AAA22356); Cry14Aa1 (Accession #AAA21516); Cry14Ab1 (Accession #KC156652); Cry15Aa1 (Accession #AAA22333); Cry16Aa1 (Accession #CAA63860); Cry17Aa1 (Accession #CAA67841); Cry18Aa1 (Accession #CAA67506); Cry18Ba1 (Accession #AAF8%67); Cry18Ca1 (Accession #AAF89668); Cry19Aa1 (Accession #CAA68875); Cry19Ba1 (Accession #BAA32397); Cry19Ca1 (Accession #AFM37572); Cry20Aa1 (Accession #AAB93476); Cry20Ba1 (Accession #ACS93601); Cry20Ba2 (Accession #KC156694); Cry20-like (Accession #GQ144333); Cry21 Aa1 (Accession #132932), Cry21Aa2 (Accession #166477); Cry21Ba1 (Accession #BAC06484); Cry21Ca1 (Accession #JF521577); Cry21Ca2 (Accession #KC156687); Cry21Da1 (Accession #JF521578); Cry22Aa1 (Accession #134547); Cry22Aa2 (Accession #CAD43579); Cry22Aa3 (Accession #ACD93211); Cry22Ab1 (Accession #AAK50456); Cry22Ab2 (Accession #CAD43577); Cry22Ba1 (Accession #CAD43578); Cry22Bb1 (Accession #KC156672); Cry23Aa1 (Accession #AAF76375); Cry24Aa1 (Accession #AAC61891); Cry24Ba1 (Accession #BAD32657); Cry24Ca1 (Accession #CAJ43600); Cry25Aa1 (Accession #AAC61892); Cry26Aa1 (Accession #AAD25075); Cry27Aa1 (Accession #BAA82796); Cry28Aa1 (Accession #AAD24189); Cry28Aa2 (Accession #AAG00235); Cry29Aa1 (Accession #CAC80985); Cry30Aa1 (Accession #CAC80986); Cry30Ba1 (Accession #BAD00052); Cry30Ca1 (Accession #BAD67157); Cry30Ca2 (Accession #ACU24781); Cry30Da1 (Accession #EF095955); Cry30db1 (Accession #BAE80088); Cry30Ea1 (Accession #ACC95445); Cry30Ea2 (Accession #FJ499389); Cry30Fa1 (Accession #ACI22625); Cry30Ga1 (Accession #ACG60020); Cry30Ga2 (Accession #HQ638217); Cry31Aa1 (Accession #BAB11757); Cry31Aa2 (Accession #AAL87458); Cry31 Aa3 (Accession #BAE79808); Cry31 Aa4 (Accession #BAF32571); Cry31 Aa5 (Accession #BAF32572); Cry31Aa6 (Accession #BA144026); Cry31Ab1 (Accession #BAE79809); Cry31 Ab2 (Accession #BAF32570); Cry31Ac1 (Accession #BAF34368); Cry31Ac2 (Accession #AB731600); Cry31Ad1 (Accession #BA144022); Cry32Aa1 (Accession #AAG36711); Cry32Aa2 (Accession #GU063849); Cry32Ab1 (Accession #GU063850); Cry32Ba1 (Accession #BAB78601); Cry32Ca1 (Accession #BAB78602); Cry32Cb1 (Accession #KC156708); Cry32Da1 (Accession #BAB78603); Cry32Ea1 (Accession #GU324274); Cry32Ea2 (Accession #KC156686), Cry32Eb1 (Accession #KC156663), Cry32Fa1 (Accession #KC156656), Cry32Ga1 (Accession #KC156657), Cry32Ha1 (Accession #KC156661); Cry32Hb1 (Accession #KC156666), Cry32Ia1 (Accession #KC156667); Cry32Ja1 (Accession #KC156685); Cry32Ka1 (Accession #KC156688), Cry32La1 (Accession #KC156689); Cry32Ma1 (Accession #KC156690); Cry32Mb1 (Accession #KC156704); Cry32Na1 (Accession #KC156691); Cry32Oa1 (Accession #KC156703); Cry32Pa1 (Accession #KC156705); Cry32Qa1 (Accession #KC156706); Cry32Ra1 (Accession #KC156707), Cry32Sa1 (Accession #KC156709); Cry32Ta1 (Accession #KC156710), Cry32Ua1 (Accession #KC156655), Cry33Aa1 (Accession #AAL26871); Cry34Aa1 (Accession #AAG50341); Cry34Aa2 (Accession #AAK64560); Cry34Aa3 (Accession #AAT29032); Cry34Aa4 (Accession #AAT29030); Cry34Ab1 (Accession #AAG41671); Cry34Ac1 (Accession #AAG50118); Cry34Ac2 (Accession #AAK64562); Cry34Ac3 (Accession #AAT29029); Cry34Ba1 (Accession #AAK64565); Cry34Ba2 (Accession #AAT29033); Cry34Ba3 (Accession #AAT29031); Cry35Aa1 (Accession #AAG50342); Cry35Aa2 (Accession #AAK64561); Cry35Aa3 (Accession #AAT29028); Cry35Aa4 (Accession #AAT29025); Cry35Ab1 (Accession #AAG41672); Cry35Ab2 (Accession #AAK64563); Cry35Ab3 (Accession #AY536891); Cry35Ac1 (Accession #AAG50117); Cry35Ba1 (Accession #AAK64566); Cry35Ba2 (Accession #AAT29027); Cry35Ba3 (Accession #AAT29026); Cry36Aa1 (Accession #AAK64558); Cry37 Aa1 (Accession #AAF76376); Cry38Aa1 (Accession #AAK64559); Cry39Aa1 (Accession #BAB72016); Cry40Aa1 (Accession #BAB72018); Cry40Ba1 (Accession #BAC77648); Cry40Ca1 (Accession #EU381045); Cry40Da1 (Accession #ACF15199); Cry41Aa1 (Accession #BAD35157); Cry41Ab1 (Accession #BAD35163); Cry41Ba1 (Accession #HM461871); Cry41Ba2 (Accession #ZP_04099652); Cry42Aa1 (Accession #BAD35166); Cry43Aa1 (Accession #BAD15301); Cry43Aa2 (Accession #BAD95474); Cry43Ba1 (Accession #BAD15303); Cry43Ca1 (Accession #KC156676); Cry43Cb1 (Accession #KC156695); Cry43Cc1 (Accession #KC156696); Cry43-like (Accession #BAD15305); Cry44Aa (Accession #BAD08532); Cry45Aa (Accession #BAD22577); Cry46Aa (Accession #BAC79010); Cry46Aa2 (Accession #BAG68906); Cry46Ab (Accession #BAD35170); Cry47 Aa (Accession #AAY24695); Cry48Aa (Accession #CAJ18351); Cry48Aa2 (Accession #CAJ86545); Cry48Aa3 (Accession #CAJ86546); Cry48Ab (Accession #CAJ86548); Cry48Ab2 (Accession #CAJ86549); Cry49Aa (Accession #CAH56541); Cry49Aa2 (Accession #CAJ86541); Cry49Aa3 (Accession #CAJ86543); Cry49Aa4 (Accession #CAJ86544), Cry49Ab1 (Accession #CAJ86542); Cry50Aa1 (Accession #BAE86999); Cry50Ba1 (Accession #GU446675); Cry50Ba2 (Accession #GU446676), Cry51Aa1 (Accession #AB114444); Cry51Aa2 (Accession #GU570697); Cry52Aa1 (Accession #EF613489), Cry52Ba1 (Accession #FJ361760); Cry53Aa1 (Accession #EF633476); Cry53Ab1 (Accession #FJ361759); Cry54Aa1 (Accession #ACA52194), Cry54Aa2 (Accession #GQ140349); Cry54Ba1 (Accession #GU446677), Cry55Aa1 (Accession #ABW88932); Cry54Ab1 (Accession #JQ916908); Cry55Aa2 (Accession #AAE33526); Cry56Aa1 (Accession #ACU57499); Cry56Aa2 (Accession #GQ483512); Cry56Aa3 (Accession #JX025567); Cry57Aa1 (Accession #ANC87261); Cry58Aa1 (Accession #ANC87260); Cry59Ba1 (Accession #JN790647); Cry59Aa1 (Accession #ACR43758); Cry60Aa1 (Accession #ACU24782); Cry60Aa2 (Accession #EA057254); Cry60Aa3 (Accession #EEM99278); Cry60Ba1 (Accession #GU810818); Cry60Ba2 (Accession #EA057253); Cry60Ba3 (Accession #EEM99279); Cry61 Aa1 (Accession #HM035087); Cry61 Aa2 (Accession #HM132125); Cry61Aa3 (Accession #EEM19308); Cry62Aa1 (Accession #HM054509); Cry63Aa1 (Accession #BA144028); Cry64Aa1 (Accession #BAJ05397); Cry65Aa1 (Accession #HM461868); Cry65Aa2 (Accession #ZP_04123838); Cry66Aa1 (Accession #HM485581); Cry66Aa2 (Accession #ZP_04099945); Cry67Aa1 (Accession #HM485582); Cry67Aa2 (Accession #ZP_04148882); Cry68Aa1 (Accession #HQ113114); Cry69Aa1 (Accession #HQ401006); Cry69Aa2 (Accession #JQ821388); Cry69Ab1 (Accession #JN209957); Cry70Aa1 (Accession #JN646781); Cry70Ba1 (Accession #AD051070); Cry70Bb1 (Accession #EEL67276); Cry71Aa1 (Accession #JX025568); Cry72Aa1 (Accession #JX025569); Cyt1Aa (GenBank Accession Number X03182); Cyt1Ab (GenBank Accession Number X98793); Cyt1B (GenBank Accession Number U37196); Cyt2A (GenBank Accession Number Z14147); and Cyt2B (GenBank Accession Number U52043).

Examples of δ-endotoxins also include but are not limited to Cry1A proteins of U.S. Pat. Nos. 5,880,275, 7,858,849 8,530,411, 8,575,433, and 8,686,233; a DIG-3 or DIG-11 toxin (N-terminal deletion of a-helix 1 and/or a-helix 2 variants of cry proteins such as Cry1A, Cry3A) of U.S. Pat. Nos. 8,304,604, 8,304,605 and 8,476,226; Cry1B of U.S. patent application Ser. No. 10/525,318, Cry1C of U.S. Pat. No. 6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960 and 6,218,188; Cry1A/F chimeras of U.S. Pat. Nos. 7,070,982; 6,962,705 and 6,713,063); a Cry2 protein such as Cry2Ab protein of U.S. Pat. No. 7,064,249); a Cry3A protein including but not limited to an engineered hybrid insecticidal protein (eHIP) created by fusing unique combinations of variable regions and conserved blocks of at least two different Cry proteins (US Patent Application Publication Number 2010/0017914); a Cry4 protein; a Cry5 protein, a Cry6 protein; Cry8 proteins of U.S. Pat. Nos. 7,329,736, 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378,499 and 7,462,760; a Cry9 protein such as such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E and Cry9F families, including but not limited to the Cry9D protein of U.S. Pat. No. 8,802,933 and the Cry9B protein of U.S. Pat. No. 8,802,934; a Cry1 5 protein of Naimov, et al., (2008), “Applied and Environmental Microbiology,” 74:7145-7151; a Cry22, a Cry34Ab1 protein of U.S. Pat. Nos. 6,127,180, 6,624,145 and 6,340,593; a CryET33 and cryET34 protein of U.S. Pat. Nos. 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 and CryET34 homologs of US Patent Publication Number 2006/0191034, 2012/0278954, and PCT Publication Number WO 2012/139004; a Cry35Ab1 protein of U.S. Pat. Nos. 6,083,499, 6,548,291 and 6,340,593; a Cry46 protein, a Cry 51 protein, a Cry binary toxin; a TIC901 or related toxin; TIC807 of US Patent Application Publication Number 2008/0295207; ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US 2006/033867; TIC853 toxins of U.S. Pat. No. 8,513,494, AXMI-027, AXMI-036, and AXMI-038 of U.S. Pat. No. 8,236,757; AXMI-031, AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. No. 7,923,602; AXMI-018, AXMI-020 and AXMI-021 of WO 2006/083891; AXMI-010 of WO 2005/038032; AXMI-003 of WO 2005/021585; AXMI-008 of US Patent Application Publication Number 2004/0250311; AXMI-006 of US Patent Application Publication Number 2004/0216186; AXMI-007 of US Patent Application Publication Number 2004/0210965; AXMI-009 of US Patent Application Number 2004/0210964; AXMI-014 of US Patent Application Publication Number 2004/0197917; AXMI-004 of US Patent Application Publication Number 2004/0197916; AXMI-028 and AXMI-029 of WO 2006/119457; AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO 2004/074462; AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 of US Patent Application Publication Number 2011/0023184; AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI-063 and AXMI-064 of US Patent Application Publication Number 2011/0263488; AXMI-R1 and related proteins of US Patent Application Publication Number 2010/0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z and AXMI225z of WO 2011/103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230 and AXMI231 of WO 2011/103247 and U.S. Pat. No. 8,759,619; AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. 8,334,431; AXMI-001, AXMI-002, AXMI-030, AXMI-035 and AXMI-045 of US Patent Application Publication Number 2010/0298211; AXMI-066 and AXMI-076 of US Patent Application Publication Number 2009/0144852; AXMI128, AXMI130, AXMU31, AXMU33, AXMU40, AXMU41, AXMU42, AXMU43, AXM1144, AXM1146, AXMU48, AXMU49, AXMU52, AXMU53, AXMI154, AXMU55, AXMU56, AXMU57, AXMU58, AXMI162, AXMU65, AXMI166, AXMU67, AXMU68, AXM1169, AXM1170, AXMU71, AXMU72, AXMU73, AXMI174, AXMU75, AXMU76, AXMU77, AXMU78, AXMI179, AXMU80, AXMU81, AXMU82, AXMU85, AXMU86, AXMU87, AXMU88, AXMU89 of U.S. Pat. No. 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI0%, AXMI097, AXMI098, AXMI099, AXMU00, AXMU01, AXMU02, AXMU03, AXMU04, AXMU07, AXMU08, AXMU09, AXMU10, AXMU11, AXMU12, AXMU14, AXMU16, AXMU17, AXMU18, AXMU19, AXMU20, AXMU21, AXMU22, AXMU23, AXMU24, AXMU257, AXMU268, AXMU27, AXMU29, AXMU64, AXMU51, AXMU61, AXMU83, AXMU32, AXMU38, AXMU37 of US Patent Application Publication Number 2010/0005543, AXMI270 of US Patent Application Publication US20140223598, AXMI279 of US Patent Application Publication US20140223599, cry proteins such as Cry1A and Cry3A having modified proteolytic sites of U.S. Pat. No. 8,319,019; a Cry1Ac, Cry2Aa and Cry1Ca toxin protein from Bacillus thuringiensis strain VBTS 2528 of US Patent Application Publication Number 2011/0064710. Other Cry proteins are well known to one skilled in the art. See, N. Crickmore, et al., “Revision of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins,” Microbiology and Molecular Biology Reviews,” (1998) Vol 62: 807-813; see also, N. Crickmore, et al, “Bacillus thuringiensis toxin nomenclature” (2016), at www.btnomenclature.info/.

The use of Cry proteins as transgenic plant traits is well known to one skilled in the art and Cry-transgenic plants including but not limited to plants expressing Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatory approval. See, Sanahuja et al., “Bacillus thuringiensis: a century of research, development and commercial applications,” (2011) Plant Biotech Journal, April 9(3):283-300 and the CERA (2010) GM Crop Database Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington D.C. at cera-gmc.org/index.php?action=gm_crop_database, which can be accessed on the world-wide web using the “www” prefix). More than one pesticidal proteins well known to one skilled in the art can also be expressed in plants such as Vip3Ab & Cry1Fa (US2012/0317682); Cry1BE & Cry1F (US2012/0311746); Cry1CA & Cry1AB (US2012/0311745); Cry1F & CryCa (US2012/0317681); Cry1DA& Cry1BE (US2012/0331590); Cry1DA & Cry1Fa (US2012/0331589); Cry1AB & Cry1BE (US2012/0324606); Cry1Fa & Cry2Aa and Cry1I & Cry1E (US2012/0324605); Cry34Ab/35Ab and Cry6Aa (US20130167269); Cry34Ab/VCry35Ab & Cry3Aa (US20130167268); Cry1Ab & Cry1F (US20140182018); and Cry3A and Cry1Ab or Vip3Aa (US20130116170). Pesticidal proteins also include insecticidal lipases including lipid acyl hydrolases of U.S. Pat. No. 7,491,869, and cholesterol oxidases such as from Streptomyces (Purcell et al. (1993) Biochem Biophys Res Commun 15:1406-1413).

Pesticidal proteins also include VIP (vegetative insecticidal proteins) toxins. Entomopathogenic bacteria produce insecticidal proteins that accumulate in inclusion bodies or parasporal crystals (such as the aforementioned Cry and Cyt proteins), as well as insecticidal proteins that are secreted into the culture medium. Among the latter are the Vip proteins, which are divided into four families according to their amino acid identity. The Vip1 and Vip2 proteins act as binary toxins and are toxic to some members of the Coleoptera and Hemiptera. The Vip1 component is thought to bind to receptors in the membrane of the insect midgut, and the Vip2 component enters the cell, where it displays its ADP-ribosyltransferase activity against actin, preventing microfilament formation. Vip3 has no sequence similarity to Vip1 or Vip2 and is toxic to a wide variety of members of the Lepidoptera. Its mode of action has been shown to resemble that of the Cry proteins in terms of proteolytic activation, binding to the midgut epithelial membrane, and pore formation, although Vip3A proteins do not share binding sites with Cry proteins. The latter property makes them good candidates to be combined with Cry proteins in transgenic plants (Bacillus thuringiensis-treated crops [Bt crops]) to prevent or delay insect resistance and to broaden the insecticidal spectrum. There are commercially grown varieties of Bt cotton and Bt maize that express the Vip3Aa protein in combination with Cry proteins. For the most recently reported Vip4 family, no target insects have been found yet. See, Chakroun et al, “Bacterial Vegetative Insecticidal Proteins (Vip) from Entomopathogenic Bacteria,” Microbiol Mol Biol Rev. 2016 Mar. 2; 80(2). 329-50. VIPs can be found in U.S. Pat. Nos. 5,877,012, 6,107,279 6,137,033, 7,244,820, 7,615,686, and 8,237,020 and the like. Other VIP proteins are well known to one skilled in the art (see, lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html, which can be accessed on the world-wide web using the “www” prefix).

Pesticidal proteins also include toxin complex (TC) proteins, obtainable from organisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, U.S. Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have “stand alone” insecticidal activity and other TC proteins enhance the activity of the stand-alone toxins produced by the same given organism. The toxicity of a “stand-alone” TC protein (from Photorhabdus, Xenorhabdus or Paenibacillus, for example) can be enhanced by one or more TC protein “potentiators” derived from a source organism of a different genus. There are three main types of TC proteins. As referred to herein, Class A proteins (“Protein A”) are stand-alone toxins. Class B proteins (“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity of Class A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 and XptA2. Examples of Class B proteins are TcaC, TcdB, XptB1Xb and XptC1 Wi. Examples of Class C proteins are TccC, XptC1Xb and XptB1 Wi. Pesticidal proteins also include spider, snake and scorpion venom proteins. Examples of spider venom peptides include, but are not limited to lycotoxin-1 peptides and mutants thereof (U.S. Pat. No. 8,334,366).

Some currently registered PIPs are listed in Table 11. Transgenic plants have also been engineered to express dsRNA directed against insect genes (Baum, J. A. et al. (2007) Control of coleopteran insect pests through RNA interference. Nature Biotechnology 25: 1322-1326; Mao, Y. B. et al. (2007) Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nature Biotechnology 25: 1307-1313). RNA interference can be triggered in the pest by feeding of the pest on the transgenic plant. Pest feeding thus causes injury or death to the pest.

TABLE 11 List of exemplary Plant-incorporated Protectants, which can be combined with microbes of the disclosure Plant-Incorporated Company and Trade Pesticide Registration Protectants (PIPs) Names Numbers Potato Potato Cry3A Potato PC Code 006432 Naturemark 524-474 New Leaf Monsanto Cry3A & PLRV Potato Monsanto 524-498 PC Codes 006432, 006469 New Leaf Plus Corn Cry1Ab Corn Event 176 PC Code 006458 Mycogen Seeds/Dow 68467-1 Agro 66736-1 Syngenta Seeds Cry1Ab Corn Event Bt11 EPA PC Code Agrisure CB (with 67979-1 006444 OECD Unique Identifier SYN- Yieldgard) 65268-1 BTØ11-1, Attribute Insect Protected Sweet Corn Syngenta Seeds Cry1Ab Corn Event MON 801 Monsanto 524-492 Cry1Ab corn Event MON 810 PC Code Monsanto 524-489 006430 OECD Unique Identifier MON- ØØ81Ø-6 Cry1Ac Corn PC Code 006463 Dekalb Genetics c/o 69575-2 Monsanto BT-XTRA Cry1F corn Event TC1507 PC Code Mycogen Seeds/Dow 68467-2 006481 OECD Unique Identifier DAS- Agro 29964-3 Ø15Ø7-1 Pioneer Hi- Bred/Dupont moCry1F corn Event DAS-Ø6275-8 PC Mycogen Seeds/Dow 68467-4 Code 006491 OECD Unique Identifier Agro DAS-Ø6275-8 Cry9C Corn Aventis 264-669 StarLink Cry3Bb1 corn Event MON863 PC Code Monsanto 524-528 006484 YielGard RW OECD Unique Identifier MON-ØØ863-5 Cry3Bb1 corn Event MON 88017 PC Code Monsanto 524-551 006498 YieldGrad VT OECD Unique Identifier MON-88Ø17-3 Rootworm Cry34Ab1/Cry35Ab1 corn Event DAS- Mycogen Seeds/Dow 68467-5 591227-7 Agro 29964-4 PC Code 006490 Pioneer Hi- OECD Unique Identifier DAS-59122-7 Bred/Dupont Herculex Rootworm Cry34Ab1/Cry35Ab1 and Cry1F corn Pioneer Hi- 29964-17 Event 4114 Bred/Dupont PC Codes 006555, 006556 mCry3A corn Event MIR 604 Syngenta Seeds 67979-5 PC Code 006509 OECD Unique Identifier Agrisure RW SYN-IR604-8 Cry1A.105 and Cry2Ab2 corn Event MON Monsanto 524-575 89034 PC Codes 006515 and 006514 Genuity VT Double Pro Vip3Aa20 corn Event MIR 162 Syngenta Seeds 67979-14 PC Code 006599 OECD Unique Identifier Agrisure Viptera SYN-IR162-4 eCry3.1Ab corn in Event 5307 PC Code Syngenta 67979-22 016483 OECD Unique Identifier SYN- Æ53Æ7-1 Stacked Events and Seed Blend Corn MON863 × MON810 with Cry3Bb1 + Monsanto YieldGard 524-545 Cry1Ab Plus DAS-59122-7 × TC1507 with Mycogen Seeds/Dow 68467-6 Cry34Ab1/Cry35Ab1 + Cry1F Agro Pioneer Hi- 29964-5 Bred/Dupont Herculex Xtra MON 88017 × MON 810 with Cry1AB + Monsanto 524-552 Cry3Bb YieldGard VT Triple YieldGard VT Plus MIR 604 × Bt11 with mCry3A + Cry1Ab Syngenta 67979-8 Agrisure CB/RW Agrisure 3000GT Mon 89034 × Mon 88017 with Cry1A.105 + Monsanto 524-576 Cry2Ab2 + Cry3Bb1 Genuity VT Triple PRO Bt11 × MIR 162 with Cry1Ab + Vip3Aa 20 Syngenta Seeds 67979-12 Agrisure 2100 Bt11 × MIR 162 × MIR 604 with Cry1Ab + Syngenta Seeds 67979-13 Vip3Aa20 + mCry3A Agrisure 3100 MON 89034 × TC1507 × MON 88017 × Monsanto Company 524-581 DAS-59122-7 with Cry1A.105 + Cry2Ab2 + Mycogen Seeds/Dow 68467-7 Cry1F + Cry3Bb1 + Agro Cry34Ab1/Cry35Ab1 Genuity SmartStax SmartStax MON 89034 × TC1507 x MON 88017 × Monsanto Company 524-595 DAS-59122-7 Seed Blend Mycogen Seeds/Dow 68467-16 Agro Genuity SmartStax RIB Complete SmartStax Refuge Advanced; Refuge Advanced Powered by SmartStax Seed Blend of Herculex Xtra + Herculex I Pioneer Hi- 29964-6 Bred/Dupont Optimum AcreMax1 Insect Protection Seed Blend of Herculex RW + Non-Bt corn Pioneer Hi- 29964-10 Bred/Dupont Optimum AcreMax RW (Cry1F × Cry34/35 × Cry1Ab) − seed blend Pioneer Hi- 29964-11 Bred/Dupont Optimum AcreMax Xtra (Cry1F × Cry1Ab) − seed blend Pioneer Hi- 29964-12 Bred/Dupont Optimum AcreMax Insect Protection (Cry1F × mCry3A) Pioneer Hi- 29964-13 Bred/Dupont Optimum Trisect (Cry1F × Cry34/35 × Cry1Ab × mCry3A) Pioneer Hi- 29964-14 Bred/Dupont Optimum Intrasect Xtreme 59122 × MON 810 × MIR 604 (Cry34/35 × Pioneer Hi- 29964-15 Cry1Ab × mCry3A) Bred/Dupont Optimum AcreMax Xtreme (Cry1F × Pioneer Hi- 29964-16 Cry34/35 × Cry1Ab × mCry3A) − seed Bred/Dupont blend Optimum AcreMax Xtreme (seed blend) MON 810 × MIR 604 (Cry1Ab × mCry3A) Pioneer Hi- 29964-18 Bred/Dupont 1507 × MON810 × MIR 162 (Cry1F × Pioneer Hi- 29964-19 Cry1Ab × Vip 3Aa20) Bred/Dupont Optimum Intrasect Leptra 1507 × MIR 162 (Cry1F × Vip30Aa20) Pioneer Hi- 29964-20 Bred/Dupont 4114 × MON 810 × MIR 604 (Cry34/35 × Pioneer Hi- 29964-21 Cry1F × Cry1Ab × mCry3A) − seed blend Bred/Dupont 4114 × MON 810 × MIR 604 (Cry34/35 × Pioneer Hi- 29964-22 Cry1F × Cry1Ab × mCry3A) Bred/Dupont 1507 × MON810 × MIR 604 (Cry1F × Pioneer Hi- 29964-23 Cry1Ab × mCry3A) − seed blend Bred/Dupont Optimum AcreMax Trisect 1507 × MON810 × MIR 604 (Cry1F × Pioneer Hi- 29964-24 Cry1Ab × mCry3A) Bred/Dupont Optimum Intrasect Trisect 4114 × MON 810 (Cry34/35 × Cry1F × Pioneer Hi- 29964-25 Cry1Ab) Bred/Dupont 1507 × MON810 × MIR 162 (Cry1F × Pioneer Hi- 29964-26 Cry1Ab × Vip 3Aa20) − seed blend Bred/Dupont Optimum AcreMax Leptra SmartStax Intermediates (8 products) Monsanto 524-583, 524-584, 524- 586, 524-587, 524- 588, 524-589, 524-590 MON 89034 × 1507 (Cry1A.105 × Monsanto 524-585 Cry2Ab2 × Cry1F) Genuity Power-Core MON 89034 (Cry1A.105 × Cry2Ab2) − Monsanto 524-597 seed blend Genuity VT Double PRO RIB Complete MON 89034 × 88017 RIB Complete Monsanto 524-606 (Cry1A.105 × Cry2Ab2 × Cry3Bb1) − seed Genuity VT Triple PRO blend RIB Complete MON 89034 × 1507 (Cry1A.105 × Monsanto 524-612 Cry2Ab2 × Cry1F) − seed blend Genuity PowerCore RIB Complete Bt11 × MIR162 × 1507 (Cry1Ab × Syngenta Seeds 67979-15 Vip3Aa20 × Cry1F) Agrisure Viptera 3220 Refuge Renew Bt11 × 59122-7 × MIR 604 × 1507 (Cry1Ab × Syngenta Seeds 67979-17 Cry34/35 × mCry3A × Cry1F) Agrisure 3122 Bt11 × MIR162 × TC1507 (Cry1Ab × Syngenta Seeds 67979-19 Vip3Aa20 × Cry1F) − seed blend Agisure Viptera 3220 (E-Z Refuge) (Refuge Advanced) Bt11 × DAS 59122-7 × MIR604 × TC1507 Syngenta Seeds 67979-20 (Cry1Ab × Cry34/35 × mCry3A × Cry1F) − Agisure Viptera 3122 seed blend (E-Z Refuge) (Refuge Advanced) Bt11 × MIR 162 × MIR 604 × TC1507 × Syngenta Seeds 67979-23 5307 (Cry1Ab × Vip3Aa20 × mCry3A × Agrisure Duracade Cry1F × eCry3.1Ab) (Refuge Renew) 5222 Bt11 × MIR 604 × TC1507 × 5307 (Cry1Ab × Syngenta Seeds 67979-24 mCry3A × Cry1F × eCry3.1Ab) Agrisure Duracade (Refuge Renew) 5122 Bt11 × MIR 604 × TC1507 × 5307 (Cry1Ab × Syngenta Seeds 67979-25 mCry3A × Cry1F × eCry3.1Ab) − seed Agisure Duracade 5122 blend E-Z Refuge Bt11 × MIR 162 × MIR 604 × TC1507 × Syngenta Seeds 67979-26 5307 (Cry1Ab × Vip3Aa20 × mCry3A × Agisure Duracade 5222 Cry1F × eCry3.1Ab) − seed blend E-Z Refuge Bt11 × MIR 162 × MIR 604 × TC1507 × Syngenta Seeds 67979-27 5307 (Cry1Ab × Vip3Aa20 x mCry3A × Agrisure Duracade Cry1F × eCry3.1Ab) (Refuge Renew) 5022 MIR604 × DAS-59122-7 × TC1507 Syngenta Seeds 67979-29 (mCry3A × Cry34/35 x Cry1F) SmartStax Intermediates (8 products) Mycogen Seeds/Dow 68467-8, 68467-9, Agro 68467-10, 68467-11, 68467-13, 68467-14, 68467-15 MON 89034 × 1507 (Cry1A.105 × Mycogen Seeds/Dow 68467-12 Cry2Ab2 × Cry1F) Agro PowerCore; PowerCore Enlist MON 89034 × 1507 (Cry1A.105 × Mycogen Seeds/Dow 68467-21 Cry2Ab2 × Cry1F) − seed blend Agro PowerCore Refuge Advanced; Refuge Advanced Powered by PowerCore 1507 × MON 810 Pioneer Hi- 29964-7 Bred/Dupont Optimum Intrasect 59122 × 1507 × MON 810 Pioneer Hi- 29964-8 Bred/Dupont 59122 × MON 810 Pioneer Hi- 29964-9 Bred/Dupont Cotton Cry1Ac Cotton Monsanto 524-478 BollGard Cry1Ac and Cry2Ab2 in Event 15985 Monsanto 524-522 Cotton PC Codes 006445, 006487 BollGard II Bt cotton Event MON531 with Cry1Ac Monsanto 524-555 (breeding nursery use only) Bt cotton Event MON15947 with Cry2Ab2 Monsanto 524-556 (breeding nursery use only) COT102 × MON 15985 (Vip3Aa19 × Monsanto 524-613 Cry1Ac × Cry2Ab2) Bollgard III Cry1F and Cry1Ac (Events DAS-21023-5 × Mycogen Seeds/Dow 68467-3 DAS-24236-5) Cotton PC Codes 006512, Agro 006513 Widestrike Event 3006-210-23 (Cry1Ac) Mycogen Seeds/Dow 68467-17 Agro Event 281-24-236 (Cry1F) Mycogen Seeds/Dow 68467-18 Agro WideStrike × COT102 (Cry1F × Cry1Ac × Mycogen Seeds/Dow 68467-19 Vip3Aa19) Agro WideStrike 3 Vip3Aa19 and FLCry1Ab (Events Syngenta Seeds 67979-9 Cot102 × Cot67B) Cotton PC Codes 016484, (Formally VipCot) 016486 OECD Unique Identifier SYN- IR102-7 X SYN-IR67B-1 COT102 (Vip3Aa19) Syngenta Seeds 67979-18 COT67B (FLCry1Ab) Syngenta Seeds 67979-21 T304-40 (Cry1Ab) Bayer CropScience 264-1094 GHB119 (Cry2Ae) Bayer CropScience 264-1095 T304-40 × GHB119 (Cry1Ab × Cry2Ae) Bayer CropScience 264-1096 OECD Unique Identifier: BCS-GHØØ4-7 × TwinLink BCS-GHØØ5-8 Soybean Cry1Ac in Event 87701 Soybean PC Code Monsanto 524-594 006532 OECD Unique Identifier Inacta Cry1A.105 and Cry2Ab2 in Event 87751 Monsanto 524-619 Soybean PC Codes 006614, 006615 OECD Unique Identifier MON-87751-7 Cry1Ac × Cry1F in Event DAS 81419 Mycogen Seeds/Dow 68467-20 Soybean PC Codes 006527, 006528 OECD Agro Unique Identifier DAS 81419 (Cry1Ac × Cry1F)

In some embodiments, any one or more of the pesticides set forth herein may be utilized with any one or more of the microbes of the disclosure and can be applied to plants or parts thereof, including seeds.

Herbicides

As aforementioned, agricultural compositions of the disclosure, which may comprise any microbe taught herein, are sometimes combined with one or more herbicides.

Compositions comprising bacteria or bacterial populations produced according to methods described herein and/or having characteristics as described herein may further include one or more herbicides. In some embodiments, herbicidal compositions are applied to the plants and/or plant parts. In some embodiments, herbicidal compositions may be included in the compositions set forth herein, and can be applied to a plant(s) or a part(s) thereof simultaneously or in succession, with other compounds.

Herbicides include 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor, ametryn, atrazine, aminopyralid, benefin, bensulfuron, bensulide, bentazon, bicyclopyrone, bromacil, bromoxynil, butylate, carfentrazone, chlorimuron, chlorsulfuron, clethodim, clomazone, clopyralid, cloransulam, cycloate, DCPA, desmedipham, dicamba, dichlobenil, diclofop, diclosulam, diflufenzopyr, dimethenamid, diquat, diuron, DSMA, endothall, EPTC, ethalfluralin, ethofumesate, fenoxaprop, fluazifop-P, flucarbzone, flufenacet, flumetsulam, flumiclorac, flumioxazin, fluometuron, fluroxypyr, fomesafen, foramsulfuron, glufosinate, glyphosate, halosulfuron, hexazinone, imazamethabenz, imazamox, imazapic, imazaquin, imazethapyr, isoxaflutole, lactofen, linuron, MCPA, MCPB, mesotrione, metolachlor-s, metribuzin, indaziflam, metsulfuron, mol mate, MSMA, napropamide, naptalam, nicosulfuron, norflurazon, oryzalin, oxadiazon, oxyfluorfen, paraquat, pelargonic acid, pendimethalin, phenmedipham, picloram, primisulfuron, prodiamine, prometryn, pronamide, propanil, prosulfuron, pyrazon, pyrithioac, quinclorac, quizalofop, rimsulfuron, 5-metolachlor, sethoxydim, siduron, simazine, sulfentrazone, sulfometuron, sulfosulfuron, tebuthiuron, tembotrione, terbacil, thiazopyr, thifensulfuron, thiobencarb, topramezone, tralkoxydim, triallate, triasulfuron, tribenuron, triclopyr, trifluralin, and triflusulfuron.

In some embodiments, any one or more of the herbicides set forth herein may be utilized with any one or more of the plants or parts thereof set forth herein.

Herbicidal products may include CORVUS, BALANCE FLEXX, CAPRENO, DIFLEXX, LIBERTY, LAUDIS, AUTUMN SUPER, and DIFLEXX DUO.

In some embodiments, any one or more of the herbicides set forth in the below Table 12 may be utilized with any one or more of the microbes taught herein, and can be applied to any one or more of the plants or parts thereof set forth herein.

TABLE 12 List of exemplary herbicides, which can be combined with microbes of the disclosure Herbicide Group Site of Action Number Chemical Family Herbicide ACCase 1 Cyclohexanediones Sethoxydim (Poast, inhibitors Poast Plus) Clethodim (Select, Select Max, Arrow) Aryloxyphenoxypropionates Fluazifop (Fusilade DX, component in Fusion) Fenoxaprop (Puma, component in Fusion) Quizalofop (Assure II, Targa) Phenylpyrazolins Pinoxaden (Axial XL) ALS inhibitors 2 Imidazolinones Imazethapyr (Pursuit) Imazamox (Raptor) Sulfonylureas Chlorimuron (Classic) Halosulfuron (Permit, Sandea) Iodosulfuron (component in Autumn Super) Mesosulfuron (Osprey) Nicosulfuron (Accent Q) Primisulfuron (Beacon) Prosulfuron (Peak) Rimsulfuron (Matrix, Resolve) Thifensulfuron (Harmony) Tribenuron (Express) Triflusulfuron (UpBeet) Triazolopyrimidine Flumetsulam (Python) Cloransulam-methyl (FirstRate) Pyroxsulam (PowerFlex HL) Florasulam (component in Quelex) Sulfonylaminocarbonyltriazolinones Propoxycarbazone (Olympus) Thiencarbazone-methyl (component in Capreno) Microtubule 3 Dinitroanilines Trifluralin (many inhibitors (root names) inhibitors) Ethalfluralin (Sonalan) Pendimethalin (Prowl/Prowl H₂O) Benzamide Pronamide (Kerb) Synthetic auxins 4 Arylpicolinate Halauxifen (Elevore, component in Quelex) Phenoxy acetic acids 2,4-D (Enlist One, others) 2,4-DB (Butyrac 200, Butoxone 200) MCPA Benzoic acids Dicamba (Banvel, Clarity, DiFlexx, Eugenia, XtendiMax; component in Status) Pyridines Clopyralid (Stinger) Fluroxypyr (Starane Ultra) Photosystem II 5 Triazines Atrazine inhibitors Simazine (Princep, Sim- Trol) Triazinone Metribuzin (Metribuzin, others) Hexazinone (Velpar) Phenyl-carbamates Desmedipham (Betenex) Phenmedipham (component in Betamix) Uracils Terbacil (Sinbar) 6 Benzothiadiazoles Bentazon (Basagran, others) Nitriles Bromoxynil (Buctril, Moxy, others) 7 Phenylureas Linuron (Lorox, Linex) Lipid synthesis 8 Thiocarbamates EPTC (Eptam) inhibitor EPSPS inhibitor 9 Organophosphorus Glyphosate Glutamine 10 Organophosphorus Glufosinate (Liberty, synthetase Rely) inhibitor Diterpene 13 Isoxazolidinone Clomazone (Command) biosynthesis inhibitor (bleaching) Protoporphyrinogen 14 Diphenylether Acifluorfen (Ultra oxidase Blazer) inhibitors (PPO) Fomesafen (Flexstar, Reflex) Lactofen (Cobra, Phoenix) N-phenylphthalimide Flumiclorac (Resource) Flumioxazin (Valor, Valor EZ, Rowel) Aryl triazolinone Sulfentrazone (Authority, Spartan) Carfentrazone (Aim) Fluthiacet-methyl (Cadet) Pyrazoles Pyraflufen-ethyl (Vida) Pyrimidinedione Saflufenacil (Sharpen) Long-chain fatty 15 Acetamides Acetochlor (Harness, acid inhibitors Surpass NXT, Breakfree NXT, Warrant) Dimethenamid-P (Outlook) Metolachlor (Parallel) Pyroxasulfone (Zidua, Zidua SC) s-metolachlor (Dual Magnum, Dual II Magnum, Cinch) Flufenacet (Define) Specific site 16 Benzofuranes Ethofumesate (Nortron) unknown Auxin transport 19 Semicarbazone diflufenzopyr inhibitor (component in Status) Photosystem I 22 Bipyridiliums Paraquat (Gramoxone, inhibitors Parazone) Diquat (Reglone) 4-HPPD 27 Isoxazole Isoxaflutole (Balance inhibitors Pyrazole Flexx) (bleaching) Pyrazolone Pyrasulfotole Triketone (component in Huskie) Topramezone (Armezon/Impact) Bicyclopyrone (component in Acuron) Mesotrione (Callisto) Tembotrione (Laudis)

Fungicides

As aforementioned, agricultural compositions of the disclosure, which may comprise any microbe taught herein, are sometimes combined with one or more fungicides.

Compositions comprising bacteria or bacterial populations produced according to methods described herein and/or having characteristics as described herein may further include one or more fungicides. In some embodiments, fungicidal compositions may be included in the compositions set forth herein, and can be applied to a plant(s) or a part(s) thereof simultaneously or in succession, with other compounds. The fungicides include azoxystrobin, captan, carboxin, ethaboxam, fludioxonil, mefenoxam, fludioxonil, thiabendazole, thiabendaz, ipconazole, mancozeb, cyazofamid, zoxamide, metalaxyl, PCNB, metaconazole, pyraclostrobin, Bacillus subtilis strain QST 713, sedaxane, thiamethoxam, fludioxonil, thiram, tolclofos-methyl, trifloxystrobin, Bacillus subtilis strain MBI 600, pyraclostrobin, fluoxastrobin, Bacillus pumilus strain QST 2808, chlorothalonil, copper, flutriafol, fluxapyroxad, mancozeb, gludioxonil, penthiopyrad, triazole, propiconaozole, prothioconazole, tebuconazole, fluoxastrobin, pyraclostrobin, picoxystrobin, qols, tetraconazole, trifloxystrobin, cyproconazole, flutriafol, SDHI, EBDCs, sedaxane, MAXIM QUATTRO (gludioxonil, mefenoxam, azoxystrobin, and thiabendaz), RAXIL (tebuconazole, prothioconazole, metalaxyl, and ethoxylated tallow alkyl amines), and benzovindiflupyr.

In some embodiments, any one or more of the fungicides set forth herein may be utilized with any one or more of the plants or parts thereof set forth herein.

Nematicides

As aforementioned, agricultural compositions of the disclosure, which may comprise any microbe taught herein, are sometimes combined with one or more nematicides.

Compositions comprising bacteria or bacterial populations produced according to methods described herein and/or having characteristics as described herein may further include one or more nematicide. In some embodiments, nematicidal compositions may be included in the compositions set forth herein, and can be applied to a plant(s) or a part(s) thereof simultaneously or in succession, with other compounds. The nematicides may be selected from D-D, 1,3-dichloropropene, ethylene dibromide, 1,2-dibromo-3-chloropropane, methyl bromide, chloropicrin, metam sodium, dazomet, methylisothiocyanate, sodium tetrathiocarbonate, aldicarb, aldoxycarb, carbofuran, oxamyl, ethoprop, fenamiphos, cadusafos, fosthiazate, terbufos, fensulfothion, phorate, DiTera, clandosan, sincocin, methyl iodide, propargyl bromide, 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP), any one or more of the avermectins, sodium azide, furfural, Bacillus firmus, abamectrin, thiamethoxam, fludioxonil, clothiandin, salicylic acid, and benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester.

In some embodiments, any one or more of the nematicides set forth herein may be utilized with any one or more of the plants or parts thereof set forth herein.

In some embodiments, any one or more of the nematicides, fungicides, herbicides, insecticides, and/or pesticides set forth herein may be utilized with any one or more of the plants or parts thereof set forth herein.

Fertilizers, Nitrogen Stabilizers, and Urease Inhibitors

As aforementioned, agricultural compositions of the disclosure, which may comprise any microbe taught herein, are sometimes combined with one or more of a: fertilizer, nitrogen stabilizer, or urease inhibitor.

In some embodiments, fertilizers are used in combination with the methods and bacteria of the present disclosure. Fertilizers include anhydrous ammonia, urea, ammonium nitrate, and urea-ammonium nitrate (UAN) compositions, among many others. In some embodiments, pop-up fertilization and/or starter fertilization is used in combination with the methods and bacteria of the present disclosure.

In some embodiments, nitrogen stabilizers are used in combination with the methods and bacteria of the present disclosure. Nitrogen stabilizers include nitrapyrin, 2-chloro-6-(trichloromethyl) pyridine, N-SERVE 24, INSTINCT, dicyandiamide (DCD).

In some embodiments, urease inhibitors are used in combination with the methods and bacteria of the present disclosure. Urease inhibitors include N-(n-butyl)-thiophosphoric triamide (NBPT), AGROTAIN, AGROTAIN PLUS, and AGROTAIN PLUS SC. Further, the disclosure contemplates utilization of AGROTAIN ADVANCED 1.0, AGROTAIN DRI-MAXX, and AGROTAIN ULTRA.

Further, stabilized forms of fertilizer can be used. For example, a stabilized form of fertilizer is SUPER U, containing 46% nitrogen in a stabilized, urea-based granule, SUPERU contains urease and nitrification inhibitors to guard from denitrification, leaching, and volatilization. Stabilized and targeted foliar fertilizer such as NITAMIN may also be used herein.

Pop-up fertilizers are commonly used in corn fields. Pop-up fertilization comprises applying a few pounds of nutrients with the seed at planting. Pop-up fertilization is used to increase seedling vigor.

Slow- or controlled-release fertilizer that may be used herein entails: A fertilizer containing a plant nutrient in a form which delays its availability for plant uptake and use after application, or which extends its availability to the plant significantly longer than a reference ‘rapidly available nutrient fertilizer’ such as ammonium nitrate or urea, ammonium phosphate or potassium chloride. Such delay of initial availability or extended time of continued availability may occur by a variety of mechanisms. These include controlled water solubility of the material by semi-permeable coatings, occlusion, protein materials, or other chemical forms, by slow hydrolysis of water-soluble low molecular weight compounds, or by other unknown means.

Stabilized nitrogen fertilizer that may be used herein entails: A fertilizer to which a nitrogen stabilizer has been added. A nitrogen stabilizer is a substance added to a fertilizer which extends the time the nitrogen component of the fertilizer remains in the soil in the urea-N or ammoniacal-N form.

Nitrification inhibitor that may be used herein entails: A substance that inhibits the biological oxidation of ammoniacal-N to nitrate-N. Some examples include: (1) 2-chloro-6-(trichloromethyl-pyridine), common name Nitrapyrin, manufactured by Dow Chemical; (2) 4-amino-1,2,4-6-triazole-HCl, common name ATC, manufactured by Ishihada Industries; (3) 2,4-diamino-6-trichloro-methyltriazine, common name CI-1580, manufactured by American Cyanamid; (4) Dicyandiamide, common name DCD, manufactured by Showa Denko; (5) Thiourea, common name TU, manufactured by Nitto Ryuso; (6) 1-mercapto-1,2,4-triazole, common name MT, manufactured by Nippon; (7) 2-amino-4-chloro-6-methyl-pyramidine, common name AM, manufactured by Mitsui Toatsu; (8) 3,4-dimethylpyrazole phosphate (DMPP), from BASF; (9) 1-amide-2-thiourea (ASU), from Nitto Chemical Ind.; (10) Ammoniumthiosulphate (ATS); (11) 1H-1,2,4-triazole (HPLC); (12) 5-ethylene oxide-3-trichloromethyl 1,2,4-thiodiazole (Terrazole), from Olin Mathieson; (13) 3-methylpyrazole (3-MP); (14) 1-carbamoyle-3-methyl-pyrazole (CMP); (15) Neem; and (16) DMPP.

Urease inhibitor that may be used herein entails: A substance that inhibits hydrolytic action on urea by the enzyme urease. Thousands of chemicals have been evaluated as soil urease inhibitors (Kiss and Simihaian, 2002). However, only a few of the many compounds tested meet the necessary requirements of being nontoxic, effective at low concentration, stable, and compatible with urea (solid and solutions), degradable in the soil and inexpensive. They can be classified according to their structures and their assumed interaction with the enzyme urease (Watson, 2000, 2005). Four main classes of urease inhibitors have been proposed: (a) reagents which interact with the sulphydryl groups (sulphydryl reagents), (b) hydroxamates, (c) agricultural crop protection chemicals, and (d) structural analogues of urea and related compounds. N-(n-Butyl) thiophosphoric triamide (NBPT), phenylphosphorodiamidate (PPD/PPDA), and hydroquinone are probably the most thoroughly studied urease inhibitors (Kiss and Simihaian, 2002). Research and practical testing has also been carried out with N-(2-nitrophenyl) phosphoric acid triamide (2-NPT) and ammonium thiosulphate (ATS). The organo-phosphorus compounds are structural analogues of urea and are some of the most effective inhibitors of urease activity, blocking the active site of the enzyme (Watson, 2005).

Insecticidal Seed Treatments (ISTs) for Corn

Corn seed treatments normally target three spectrums of pests, nematodes, fungal seedling diseases, and insects.

Insecticide seed treatments are usually the main component of a seed treatment package. Most corn seed available today comes with a base package that includes a fungicide and insecticide. In some aspects, the insecticide options for seed treatments include PONCHO (clothianidin), CRUISER/CRUISER EXTREME (thiamethoxam) and GAUCHO (Imidacloprid). All three of these products are neonicotinoid chemistries. CRUISER and PONCHO at the 250 (0.25 mg AI/seed) rate are some of the most common base options available for corn. In some aspects, the insecticide options for treatments include CRUISER 250 thiamethoxam, CRUISER 250 (thiamethoxam) plus LUMIVIA (chlorantraniliprole), CRUISER 500 (thiamethoxam), and PONCHO VOTIVO 1250 (Clothianidin & Bacillus firmus I-1582).

Pioneer's base insecticide seed treatment package consists of CRUISER 250 with PONCHO/VOTIVO 1250 also available. VOTIVO is a biological agent that protects against nematodes.

Monsanto's products including corn, soybeans, and cotton fall under the ACCELERON treatment umbrella. Dekalb corn seed comes standard with PONCHO 250. Producers also have the option to upgrade to PONCHO/VOTIVO, with PONCHO applied at the 500 rate.

Agrisure, Golden Harvest and Garst have a base package with a fungicide and CRUISER 250. AVICTA complete corn is also available, this includes CRUISER 500, fungicide, and nematode protection. CRUISER EXTREME is another option available as a seed treatment package, however; the amounts of CRUISER are the same as the conventional CRUISER seed treatment, i.e. 250, 500, or 1250.

Another option is to buy the minimum insecticide treatment available, and have a dealer treat the seed downstream.

Commercially available ISTs for corn are listed in the below Table 13 and can be combined with one or more of the microbes taught herein.

TABLE 13 List of exemplary seed treatments, including ISTs, which can be combined with microbes of the disclosure Treatment Type Active Ingredient(s) Product Trade Name Crop F azoxystrobin DYNASTY Corn, Soybean PROTÉGÉ FL Corn F Bacillus pumilus YIELD SHIELD Corn, Soybean F Bacillus subtilis HISTICK N/T Soybean VAULT HP Corn, Soybean F Captan CAPTAN 400 Corn, Soybean CAPTAN 400-C Corny Soybean F Fludioxonil MAXIM 4FS Corn, Soybean F Hydrogen peroxide OXIDATE Soybean STOROX Soybean F ipconazole ACCELERON DC-509 Corn RANCONA 3.8 FS Corn, Soybean VORTEX Corn F mancozeb BONIDE MANCOZEB w/Zinc Corn Concentrate DITHANE 75DF RAINSHIELD Corn DITHANE DF RAINSHIELD Corn DITHANE F45 RAINSHIELD Corn DITHANE M45 Corn LESCO 4 FLOWABLE Corn MANCOZEB PENNCOZEB 4FL FLOWABLE PENNCOZEB 75DF DRY Corn FLOWABLE Corn PENNCOZEB 80WP Corn F mefenoxam APRON XL Corn, Soybean F metalaxyl ACCELERON DC-309 Corn ACCELERON DX-309 Corn, Soybean ACQUIRE Corn, Soybean AGRI STAR METALAXYL 265 Corn, Soybean ST ALLEGIANCE DRY Corn, Soybean ALLEGIANCE FL Corn, Soybean BELMONT 2.7 FS Corn, Soybean DYNA-SHIELD METALAXYL Corn, Soybean SEBRING 2.65 ST Corn, Soybean SEBRING 318 FS Corn, Soybean SEBRING 480 FS Corn, Soybean VIREO MEC Soybean F pyraclostrobin ACCELERON DX-109 Soybean STAMINA Corn F Streptomyces MYCOSTOP Corn, Soybean griseoviridis F Streptomyces lydicus ACTINOGROW ST Corn, Soybean F tebuconazole AMTIDE TEBU 3.6F Corn SATIVA 309 FS Corn SATIVA 318 FS Corn TEBUSHA 3.6FL Corn TEBUZOL 3.6F Corn F thiabendazole MERTECT 340-F Soybean F thiram 42-S THIRAM Corn, Soybean FLOWSAN Corn, Soybean SIGNET 480 FS Corn, Soybean F Trichoderma T-22 HC Corn, Soybean harzianum Rifai F trifloxystrobin ACCELERON DX-709 Corn TRILEX FLOWABLE Corn, soybean I chlorpyrifos LORSBAN 50W in water soluble Corn packets I clothianidin ACCELERON IC-609 Corn NIPSIT INSIDE Corn, Soybean PONCHO 600 Corn I imidacloprid ACCELERON IX-409 Corn AGRI STAR MACHO 600 ST Corn, Soybean AGRISOLUTIONS NITRO Corn, Soybean SHIELD ATTENDANT 600 Corn, Soybean AXCESS Corn, Soybean COURAZE 2F Soybean DYNA-SHIELD Corn, Soybean IMIDACLOPRID 5 GAUCHO 480 FLOWABLE Corn, Soybean GAUCHO 600 FLOWABLE Corn, Soybean GAUCHO SB FLOWABLE Corn, Soybean NUPRID 4.6F PRO Soybean SENATOR 600 FS Corn, Soybean I thiamethoxam CRUISER 5FS Corn, Soybean N abamectin AVICTA 500 FS Corn, Soybean N Bacillus firmus VOTIVO FS Soybean P cytokinin SOIL X-CYTO Soybean X-CYTE Soybean P harpin alpha beta ACCELERON HX-209 Corn, Soybean protein N-HIBIT GOLD CST Corn, Soybean N-HIBIT HX-209 Corn, Soybean P indole butyric acid KICKSTAND PGR Corn, Soybean I, N thiamethoxam, AVICTA DUO CORN Corn abamectin AVICTA DUO 250 I, F clothianidin, PONCHO VOTIVO Corn, Soybean Bacillus firmus F, F carboxin, captan ENHANCE Soybean I, F permethrin, carboxin KERNEL GUARD SUPREME Corn, Soybean F, F carboxin, thiram VITAFLO 280 Corn, Soybean F, F mefenoxam, fludioxonil MAXIM XL Corn, Soybean WARDEN RTA Soybean APRON MAXX RFC APRON MAXX RTA + MOLY APRON MANX RTA I, F imidacloprid, metalaxyl AGRISOLUTIONS CONCUR Corn F, F metalaxyl, ipconazole RANCONA SUMMIT Soybean RANCONA XXTRA F, F thiram, metalaxyl PROTECTOR-L-ALLEGIANCE Soybean F, F trifloxystrobin, TRILEX AL Soybean metalaxyl TRILEX 2000 P, P, P cytokinin, gibberellic STIMULATE YIELD Corn, Soybean acid, indole butyric acid ENHANCER ASCEND F, F, I mefenoxam, CRUISERMAXX PLUS Soybean fludioxonil, thiamethoxam F, F, F captan, carboxin, BEAN GUARD/ALLEGIANCE Soybean metalaxyl F, F, I captan, carboxin, ENHANCE AW Soybean imidacloprid F, F, I carboxin, LATITUDE Corn, Soybean metalaxyl, imidacloprid F, F, F metalaxyl, STAMINA F3 HL Corn pyraclostrobin, triticonazole F, F, F, I azoxystrobin, CRUISER EXTREME Corn fludioxonil, mefenoxam, thiamethoxam F, F, F, F, F azoxystrobin, MAXIM QUATTRO Corn fludioxonil, mefenoxam, thiabendazole I Chlorantraniliprole LUMIVIA Corn F = Fungicide; I = Insecticide; N = Nematicide; P = Plant Growth Regulator

Application of Bacterial Populations on Crops

The composition of the bacteria or bacterial population described herein can be applied in furrow, in talc, or as seed treatment. The composition can be applied to a seed package in bulk, mini bulk, in a bag, or in talc.

The planter can plant the treated seed and grows the crop according to conventional ways, twin row, or ways that do not require tilling. The seeds can be distributed using a control hopper or an individual hopper. Seeds can also be distributed using pressurized air or manually. Seed placement can be performed using variable rate technologies. Additionally, application of the bacteria or bacterial population described herein may be applied using variable rate technologies. In some examples, the bacteria can be applied to seeds of corn, soybean, canola, sorghum, potato, rice, vegetables, cereals, pseudocereals, and oilseeds. Examples of cereals may include barley, fonio, oats, palmer's grass, lye, pearl millet, sorghum, spelt, teff, triticale, and wheat. Examples of pseudocereals may include breadnut, buckwheat, cattail, chia, flax, grain amaranth, hanza, quinoa, and sesame. In some examples, seeds can be genetically modified organisms (GMO), non-GMO, organic or conventional.

Additives such as micro-fertilizer, PGR, herbicide, insecticide, and fungicide can be used additionally to treat the crops. Examples of additives include crop protectants such as insecticides, nematicides, fungicide, enhancement agents such as colorants, polymers, pelleting, priming, and disinfectants, and other agents such as inoculant, PGR, softener, and micronutrients. PGRs can be natural or synthetic plant hormones that affect root growth, flowering, or stem elongation. PGRs can include auxins, gibberellins, cytokinins, ethylene, and abscisic acid (ABA).

The composition can be applied in furrow in combination with liquid fertilizer. In some examples, the liquid fertilizer may be held in tanks. NPK fertilizers contain macronutrients of sodium, phosphorous, and potassium.

The composition may improve plant traits, such as promoting plant growth, maintaining high chlorophyll content in leaves, increasing fruit or seed numbers, and increasing fruit or seed unit weight. Methods of the present disclosure may be employed to introduce or improve one or more of a variety of desirable traits. Examples of traits that may introduced or improved include: root biomass, root length, height, shoot length, leaf number, water use efficiency, overall biomass, yield, fruit size, grain size, photosynthesis rate, tolerance to drought, heat tolerance, salt tolerance, tolerance to low nitrogen stress, nitrogen use efficiency, resistance to nematode stress, resistance to a fungal pathogen, resistance to a bacterial pathogen, resistance to a viral pathogen, level of a metabolite, modulation in level of a metabolite, proteome expression. The desirable traits, including height, overall biomass, root and/or shoot biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, seed/fruit number or mass, plant grain or fruit yield, leaf chlorophyll content, photosynthetic rate, root length, or any combination thereof, can be used to measure growth, and compared with the growth rate of reference agricultural plants (e.g., plants without the introduced and/or improved traits) grown under identical conditions. In some examples, the desirable traits, including height, overall biomass, root and/or shoot biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, seed/fruit number or mass, plant grain or fruit yield, leaf chlorophyll content, photosynthetic rate, root length, or any combination thereof, can be used to measure growth, and compared with the growth rate of reference agricultural plants (e.g., plants without the introduced and/or improved traits) grown under similar conditions.

An agronomic trait to a host plant may include, but is not limited to, the following: altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, and altered seed protein composition, chemical tolerance, cold tolerance, delayed senescence, disease resistance, drought tolerance, ear weight, growth improvement, health enhancement, heat tolerance, herbicide tolerance, herbivore resistance improved nitrogen fixation, improved nitrogen utilization, improved root architecture, improved water use efficiency, increased biomass, increased root length, increased seed weight, increased shoot length, increased yield, increased yield under water-limited conditions, kernel mass, kernel moisture content, metal tolerance, number of ears, number of kernels per ear, number of pods, nutrition enhancement, pathogen resistance, pest resistance, photosynthetic capability improvement, salinity tolerance, stay-green, vigor improvement, increased dry weight of mature seeds, increased fresh weight of mature seeds, increased number of mature seeds per plant, increased chlorophyll content, increased number of pods per plant, increased length of pods per plant, reduced number of wilted leaves per plant, reduced number of severely wilted leaves per plant, and increased number of non-wilted leaves per plant, a detectable modulation in the level of a metabolite, a detectable modulation in the level of a transcript, and a detectable modulation in the proteome, compared to an isoline plant grown from a seed without said seed treatment formulation.

In some cases, plants are inoculated with bacteria or bacterial populations that are isolated from the same species of plant as the plant element of the inoculated plant. For example, a bacteria or bacterial population that is normally found in one variety of Zea mays (corn) is associated with a plant element of a plant of another variety of Zea mays that in its natural state lacks said bacteria and bacterial populations. In one embodiment, the bacteria and bacterial populations is derived from a plant of a related species of plant as the plant element of the inoculated plant. For example, a bacteria and bacterial populations that is normally found in Zea diploperennis Iltis et al., (diploperennial teosinte) is applied to a Zea mays (corn), or vice versa. In some cases, plants are inoculated with bacteria and bacterial populations that are heterologous to the plant element of the inoculated plant. In one embodiment, the bacteria and bacterial populations is/are derived from a plant of another species. For example, a bacteria and bacterial population that is normally found in dicots is applied to a monocot plant (e.g., inoculating corn with a soybean-derived bacteria and bacterial populations), or vice versa. In other cases, the bacteria and bacterial populations to be inoculated onto a plant is derived from a related species of the plant that is being inoculated. In one embodiment, the bacteria and bacterial populations is derived from a related taxon, for example, from a related species. The plant of another species can be an agricultural plant. In another embodiment, the bacteria and bacterial populations is part of a designed composition inoculated into any host plant element.

In some examples, the bacteria or bacterial population is exogenous wherein the bacteria and bacterial population is isolated from a different plant than the inoculated plant. For example, in one embodiment, the bacteria or bacterial population can be isolated from a different plant of the same species as the inoculated plant. In some cases, the bacteria or bacterial population can be isolated from a species related to the inoculated plant.

In some examples, the bacteria and bacterial populations described herein are capable of moving from one tissue type to another. For example, the present disclosure's detection and isolation of bacteria and bacterial populations within the mature tissues of plants after coating on the exterior of a seed demonstrates their ability to move from seed exterior into the vegetative tissues of a maturing plant. Therefore, in one embodiment, the population of bacteria and bacterial populations is capable of moving from the seed exterior into the vegetative tissues of a plant. In one embodiment, the bacteria and bacterial populations that is coated onto the seed of a plant is capable, upon germination of the seed into a vegetative state, of localizing to a different tissue of the plant. For example, bacteria and bacterial populations can be capable of localizing to any one of the tissues in the plant, including: the root, adventitious root, seminal 5 root, root hair, shoot, leaf, flower, bud, tassel, meristem, pollen, pistil, ovaries, stamen, fruit, stolon, rhizome, nodule, tuber, trichome, guard cells, hydathode, petal, sepal, glume, rachis, vascular cambium, phloem, and xylem. In one embodiment, the bacteria and bacterial populations is capable of localizing to the root and/or the root hair of the plant. In another embodiment, the bacteria and bacterial populations is capable of localizing to the photosynthetic tissues, for example, leaves and shoots of the plant. In other cases, the bacteria and bacterial populations is localized to the vascular tissues of the plant, for example, in the xylem and phloem. In still another embodiment, the bacteria and bacterial populations is capable of localizing to the reproductive tissues (flower, pollen, pistil, ovaries, stamen, fruit) of the plant. In another embodiment, the bacteria and bacterial populations is capable of localizing to the root, shoots, leaves and reproductive tissues of the plant. In still another embodiment, the bacteria and bacterial populations colonizes a fruit or seed tissue of the plant. In still another embodiment, the bacteria and bacterial populations is able to colonize the plant such that it is present in the surface of the plant (i.e., its presence is detectably present on the plant exterior, or the episphere of the plant). In still other embodiments, the bacteria and bacterial populations is capable of localizing to substantially all, or all, tissues of the plant. In certain embodiments, the bacteria and bacterial populations is not localized to the root of a plant. In other cases, the bacteria and bacterial populations is not localized to the photosynthetic tissues of the plant.

The effectiveness of the compositions can also be assessed by measuring the relative maturity of the crop or the crop heating unit (CHU). For example, the bacterial population can be applied to corn, and corn growth can be assessed according to the relative maturity of the corn kernel or the time at which the corn kernel is at maximum weight. The crop heating unit (CHU) can also be used to predict the maturation of the corn crop. The CHU determines the amount of heat accumulation by measuring the daily maximum temperatures on crop growth.

In examples, bacterial may localize to any one of the tissues in the plant, including: the root adventitious root, seminal root, root hair, shoot, leaf, flower, bud tassel, meristem, pollen, pistil, ovaries, stamen, fruit, stolon, rhizome, nodule, tuber, trichome, guard cells, hydathode, petal, sepal, glume, rachis, vascular cambium, phloem, and xylem. In another embodiment, the bacteria or bacterial population is capable of localizing to the photosynthetic tissues, for example, leaves and shoots of the plant. In other cases, the bacteria and bacterial populations is localized to the vascular tissues of the plant, for example, in the xylem and phloem. In another embodiment, the bacteria or bacterial population is capable of localizing to reproductive tissues (flower, pollen, pistil, ovaries, stamen, or fruit) of the plant. In another embodiment the bacteria and bacterial populations is capable of localizing to the root shoots, leaves and reproductive tissues of the plant. In another embodiment the bacteria or bacterial population colonizes a fruit or seed tissue of the plant. In still another embodiment the bacteria or bacterial population is able to colonize the plant such that it is present in the surface of the plant. In another embodiment the bacteria or bacterial population is capable of localizing to substantially all, or all, tissues of the plant. In certain embodiments, the bacteria or bacterial population is not localized to the root of a plant. In other cases, the bacteria and bacterial populations is not localized to the photosynthetic tissues of the plant.

The effectiveness of the bacterial compositions applied to crops can be assessed by measuring various features of crop growth including, but not limited to, planting rate, seeding vigor, root strength, drought tolerance, plant height, dry down, and test weight.

Plant Species

The methods and bacteria described herein are suitable for any of a variety of plants, such as plants in the genera Hordeum, Oryza, Zea, and Triticeae. Other non-limiting examples of suitable plants include mosses, lichens, and algae. In some cases, the plants have economic, social and/or environmental value, such as food crops, fiber crops, oil crops, plants in the forestry or pulp and paper industries, feedstock for biofuel production and/or ornamental plants. In some examples, plants may be used to produce economically valuable products such as a grain, a flour, a starch, a syrup, a meal, an oil, a film, a packaging, a nutraceutical product, a pulp, an animal feed, a fish fodder, a bulk material for industrial chemicals, a cereal product, a processed human-food product, a sugar, an alcohol, and/or a protein. Non-limiting examples of crop plants include maize, rice, wheat, barley, sorghum, millet, oats, rye triticale, buckwheat sweet corn, sugar cane, onions, tomatoes, strawberries, and asparagus. In some embodiments, the methods and bacteria described herein are suitable for any of a variety of transgenic plants, non-transgenic plants, and hybrid plants thereof.

In some examples, plants that may be obtained or improved using the methods and composition disclosed herein may include plants that are important or interesting for agriculture, horticulture, biomass for the production of biofuel molecules and other chemicals, and/or forestry. Some examples of these plants may include pineapple, banana, coconut, lily, grasspeas and grass; and dicotyledonous plants, such as, for example, peas, alfalfa, tomatillo, melon, chickpea, chicory, clover, kale, lentil, soybean, tobacco, potato, sweet potato, radish, cabbage, rape, apple trees, grape, cotton, sunflower, thale cress, canola, citrus (including orange, mandarin, kumquat, lemon, lime, grapefruit, tangerine, tangelo, citron, and pomelo), pepper, bean, lettuce, Panicum virgatum (switch), Sorghum bicolor (sorghum, Sudan), Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays (corn), Glycine max (soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet), Pennisetum glaucum (pearl millet), Panicum spp. Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp., Secale cereale (rye), Salix spp. (willow), Eucalyptus spp. (eucalyptus), Triticosecale spp. (triticum-25 wheat X rye), Bamboo, Carthamus tinctorius (safflower), Jatropha curcas (Jatropha), Ricinus communis (castor), Elaeis guineensis (oil palm), Phoenix dactylifera (date palm), Archontophoenix cunninghamiana (king palm), Syagrus romanzoffiana (queen palm), Linum usitatissimum (flax), Brassica juncea, Manihot esculenta (cassaya), Lycopersicon esculentum (tomato), Lactuca saliva (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato), Brassica oleracea (broccoli, cauliflower, brussel sprouts), Camellia sinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa), Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion), Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima (squash), Cucurbita moschata (squash), Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), Solanum melongena (eggplant), Papaver somniferum (opium poppy), Papaver orientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabis saliva, Camptotheca acuminate, Catharanthus roseus, Vinca rosea, Cinchona officinalis, Coichicum autumnale, Veratrum californica, Digitalis lanata, Digitalis purpurea, Dioscorea 5 spp., Andrographis paniculata, Atropa belladonna, Datura stomonium, Berberis spp., Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca, Galanthus wornorii, Scopolia spp., Lycopodium serratum (Huperzia serrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp., Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis, Chrysanthemum parthenium, Coleus forskohlii, Tanacetum parthenium, Parthenium argentatum (guayule), Hevea spp. (rubber), Mentha spicata (mint), Mentha piperita (mint), Bixa orellana, Alstroemeria spp., Rosa spp. (rose), Dianthus caryophyllus (carnation), Petunia spp. (petunia), Poinsettia pulcherrima (poinsettia), Nicotiana tabacum (tobacco), Lupinus albus (lupin), Uniola paniculata (oats), Hordeum vulgare (barley), and Lolium spp. (rye).

In some examples, a monocotyledonous plant may be used. Monocotyledonous plants belong to the orders of the Alismatales, Arales, Arecales, Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales, Hydrocharitales, Juncales, Lilliales, Najadales, Orchidales, Pandanales, Poales, Restionales, Triuridales, Typhales, and Zingiberales. Plants belonging to the class of the Gymnospermae are Cycadales, Ginkgoales, Gnetales, and Pinales. In some examples, the monocotyledonous plant can be selected from the group consisting of a maize, rice, wheat, barley, and sugarcane.

In some examples, a dicotyledonous plant may be used, including those belonging to the orders of the Aristochiales, Asterales, Batales, Campanulales, Capparales, CaryophyHales, Casuarinales, Celastrales, Comales, Diapensales, Dilleniales, Dipsacales, Ebenales, Ericales, Eucomiales, Euphorbiales, Fabales, Fagales, Gentianales, Geraniales, Halora gales, Hamamelidales, Middles, Juglandales, Lamiales, Laurales, Lecythidales, Leitneriales, Magniolales, Malvales, Myricales, Myrtales, Nymphaeales, Papeverales, Piperales, Plantaginales, Plumb aginales, Podostemales, Polemoniales, Polygalales, Polygonales, Primulales, Proteales, Rafflesiales, Ranunculales, Rhamnales, Rosales, Rubiales, Salicales, Santales, Sapindales, Sarraceniaceae, Scrophulariales, Theales, Trochodendrales, Umbellales, Urticales, and Violates. In some examples, the dicotyledonous plant can be selected from the group consisting of cotton, soybean, pepper, and tomato.

In some cases, the plant to be improved is not readily amenable to experimental conditions. For example, a crop plant may take too long to grow enough to practically assess an improved trait serially over multiple iterations. Accordingly, a First plant from which bacteria are initially isolated, and/or the plurality of plants to which genetically manipulated bacteria are applied may be a model plant, such as a plant more amenable to evaluation under desired conditions. Non-limiting examples of model plants include Setaria, Brachypodium, and Arabidopsis. Ability of bacteria isolated according to a method of the disclosure using a model plant may then be applied to a plant of another type (e.g. a crop plant) to confirm conferral of the improved trait.

Traits that may be improved by the methods disclosed herein include any observable characteristic of the plant, including, for example, growth rate, height, weight, color, taste, smell changes in the production of one or more compounds by the plant (including for example, metabolites, proteins, drugs, carbohydrates, oils, and any other compounds). Selecting plants based on genotypic information is also envisaged (for example, including the pattern of plant gene expression in response to the bacteria, or identifying the presence of genetic markers, such as those associated with increased nitrogen fixation). Plants may also be selected based on the absence, suppression or inhibition of a certain feature or trait (such as an undesirable feature or trait) as opposed to the presence of a certain feature or trait (such as a desirable feature or trait).

Non-Genetically Modified Maize

The methods and bacteria described herein are suitable for any of a variety of non-genetically modified maize plants or part thereof. And in some aspects the corn is organic. Furthermore, the methods and bacteria described herein are suitable for any of the following non-genetically modified hybrids, varieties, lineages, etc. In some embodiments, corn varieties generally fall under six categories: sweet corn, flint corn, popcorn, dent corn, pod corn, and flour corn.

Sweet Corn

Yellow su varieties include Earlivee, Early Sunglow, Sundance, Early Golden Bantam, lochief, Merit, Jubilee, and Golden Cross Bantam. White su varieties include True Platinum, Country Gentleman, Silver Queen, and Stowell's Evergreen. Bicolor su varieties include Sugar & Gold, Quickie, Double Standard, Butter & Sugar, Sugar Dots, Honey & Cream. Multicolor su varieties include Hookers, Triple Play, Painted Hill, Black Mexican/Aztec.

Yellow se varieties include Buttergold, Precocious, Spring Treat, Sugar Buns, Colorow, Kandy King, Bodacious R/M, Tuxedo, Incredible, Merlin, Miracle, and Kandy Korn EH. White se varieties include Spring Snow, Sugar Pearl, Whiteout, Cloud Nine, Alpine, Silver King, and Argent. Bicolor se varieties include Sugar Baby, Fleet, Bon Jour, Trinity, Bi-Licious, Temptation, Luscious, Ambrosia, Accord, Brocade, Lancelot, Precious Gem, Peaches and Cream Mid EH, and Delectable R/M. Multicolor se varieties include Ruby Queen.

Yellow sh2 varieties include Extra Early Super Sweet, Takeoff, Early Xtra Sweet, Raveline, Summer Sweet Yellow, Krispy King, Garrison, Illini Gold, Challenger, Passion, Excel, Jubilee SuperSweet, Illini Xtra Sweet, and Crisp 'N Sweet. White sh2 varieties include Summer Sweet White, Tahoe, Aspen, Treasure, How Sweet It Is, and Camelot Bicolor sh2 varieties include Summer Sweet Bicolor, Radiance, Honey 'N Pearl, Aloha, Dazzle, Hudson, and Phenomenal.

Yellow sy varieties include Applause, Inferno, Honeytreat, and Honey Select White sy varieties include Silver Duchess, Cinderella, Mattapoisett, Avalon, and Captivate. Bicolor sy varieties include Pay Dirt, Revelation, Renaissance, Charisma, Synergy, Montauk, Kristine, Serendipity-Providence, and Cameo.

Yellow augmented supersweet varieties include Xtra-Tender 1ddA, Xtra-Tender 11dd, Mirai 131Y, Mirai 130Y, Vision, and Mirai 002. White augmented supersweet varieties include Xtra-Tender 3dda, Xtra-Tender 31dd, Mirai 421W, XTH 3673, and Devotion. Bicolor augmented supersweet varieties include Xtra-Tender 2dda, Xtra-Tender 21dd, Kickoff XR, Mirai 308BC, Anthem XR, Mirai 336BC, Fantastic XR, Triumph, Mirai 301BC, Stellar, American Dream, Mirai 350BC, and Obsession.

Flint Corn

Flint corn varieties include Bronze-Orange, Candy Red Flint, Floriani Red Flint, Glass Gem, Indian Ornamental (Rainbow), Mandan Red Flour, Painted Mountain, Petmecky, Cherokee White Flour,

PopCorn

Pop corn varieties include Monarch Butterfly, Yellow Butterfly, Midnight Blue, Ruby Red, Mixed Baby Rice, Queen Mauve, Mushroom Flake, Japanese Hull-less, Strawberry, Blue Shaman, Miniature Colored, Miniature Pink, Pennsylvania Dutch Butter Flavor, and Red Strawberry.

Dent Corn

Dent corn varieties include Bloody Butcher, Blue Clarage, Ohio Blue Clarage, Cherokee White Eagle, Hickory Cane, Hickory King, Jellicorse Twin, Kentucky Rainbow, Daymon Morgan's Knt. Butcher, Learning, Learning's Yellow, McCormack's Blue Giant, Neal Paymaster, Pungo Creek Butcher, Reid's Yellow Dent, Rotten Clarage, and Tennessee Red Cob.

In some embodiments, corn varieties include P1618W, P1306W, P1345, P1151, P1197, P0574, P0589, and P0157. W=white corn.

In some embodiments, the methods and bacteria described herein are suitable for any hybrid of the maize varieties setforth herein.

Genetically Modified Maize

The methods and bacteria described herein are suitable for any of a hybrid, variety, lineage, etc. of genetically modified maize plants or part thereof.

Furthermore, the methods and bacteria described herein are suitable for any of the following genetically modified maize events, which have been approved in one or more countries. 32138 (32138 SPT Maintainer), 3272 (ENOGEN), 3272×Bt11, 3272×bt11×GA21, 3272×Bt11×MIR604, 3272×Bt11×MIR604×GA21, 3272×Bt11×MIR604×TC1507×5307×GA21, 3272×GA21, 3272×MIR604, 3272×MIR604×GA21, 4114, 5307 (AGRISURE Duracade), 5307×GA21, 5307×MIR604×Bt11×TC1507×GA21 (AGRISURE Duracade 5122), 5307×MIR604×Bt11×TC1507×GA21×MIR162 (AGRISURE Duracade 5222), 59122 (HERCULEX RW), 59122×DAS40278, 59122×GA21, 59122×MIR604, 59122×MIR604×GA21, 59122×MIR604×TC1507, 59122×MIR604×TC1507×GA21, 59122×MON810, 59122×MON810×MIR604, 59122×MON810×NK603, 59122×MON810×NK603×MIR604, 59122×MON88017, 59122×MON88017×DAS40278, 59122×NK603 (Herculex RW ROUNDUP READY 2), 59122×NK603×MIR604, 59122×TC1507×GA21, 676, 678, 680, 3751 IR, 98140, 98140×59122, 98140×TC1507, 98140×TC1507×59122, Bt10 (Bt10), Bt11 [X4334CBR, X4734CBR] (AGRISURE CB/LL), Bt11×5307, Bt11×5307×GA21, Bt11×59122×MIR604, Br11×59122×MIR604×GA21, Bt11×59122×MIR604×TC1507, M53, M56, DAS-59122-7, Bt11×59122×MIR604×TC1507×GA21, Bt11×59122×TC1507, TC1507×DAS-59122-7, Bt11×59122×TC1507×GA21, Bt11×GA21 (AGRISURE GT/CB/LL), Bt11×MIR162 (AGRISURE Viptera 2100), BT11×MIR162×5307, Bt11×MIR162×5307×GA21, Bt11×MIR162×GA21 (AGRISURE Viptera 3110), Bt11×MIR162×MIR604 (AGRISURE Viptera 3100), Bt11×MIR162×MIR604×5307, Bt11×MIR162×MIR604×5307×GA21, Bt11×MIR162×MIR604×GA21 (AGRISURE Viptera 3111/AGRISURE Viptera 4), Bt11, MIR162×MIR604×MON89034×5307×GA21, Bt11×MIR162×MIR604×TC1507, Bt11×MIR162×MIR604×TC1507×5307, Bt11×MIR162×MIR604×TC1507×GA21, Bt11×MIR162×MON89034, Bt11×MIR162×MON89034×GA21, Bt11×MIR162×TC1507, Bt11×MIR162×TC1507×5307, Bt11×MIR162×TC1507×5307×GA21, Bt11×MR162×TC1507×GA21 (AGRISURE Viptera 3220), BT11×MIR604 (Agrisure BC/LL/RW), Bt11×MIR604×5307, Bt11×MIR604×5307×GA21, Bt11×MIR604×GA21, Bt11×MIR604×TC1507, Bt11×MIR604×TC1507×5307, Bt11×MIR604×TC1507×GA21, Bt11×MON89Ø34×GA21, Bt11×TC1507, Bt11×TC1507×5307, Bt11×TC1507×GA21, Bt176 [176] (NaturGard KnockOut/Maximizer), BVLA430101, CBH-351 (STARLINK Maize), DAS40278 (ENLIST Maize), DAS40278×NK603, DBT418 (Bt Xtra Maize), DLL25 [B16], GA21 (ROUNDUP READY Maize/AGRISURE GT), GA21×MON810 (ROUNDUP READY Yieldgard Maize), GA21×T25, HCEM485, LY038 (MAVERA Maize), LY038×MON810 (MAVERA Yieldgard Maize), MIR162 (AGRISURE Viptera), MIR162×5307, MIR162×5307×GA21, MIR162×GA21, MIR162×MIR604, MIR162×MIR604×5307, MIR162×MIR604×5307×GA21, MIR162×MIR604×GA21, MIR162×MIR604×TC1507×5307, MIR162×MIR604×TC1507×5307×GA21, MIR162×MIR604×TC1507×GA21, MIR162×MON89034, MIR162×NK603, MIR162×TC1507, MIR162×TC1507×5307, MIR162×TC1507×5307×GA21, MIR162×TC1507×GA21, MIR604 (AGRISURE RW), MIR604×5307, MIR604×5307×GA21, MIR604×GA21 (AGRISURE GT/RW), MIR604×NK603, MIR604×TC1507, MIR604×TC1507×5307, MIR604×TC1507×5307×GA21, MIR604×TC1507×GA21, MON801 [MON80100], MON802, MON809, MON810 (YIELDGARD, MAIZEGARD), MON810×MIR162, MON810×MIR162×NK603, MON810×MIR604, MON810×MON88017 (YIELDGARD VT Triple), MON810×NK603×MIR604, MON832 (ROUNDUP READY Maize), MON863 (YIELDGARD Rootworm RW, MAXGARD), MON863×MON810 (YIELDGARD Plus), MON863×MON810×NK603 (YIELDGARD Plus with RR), MON863×NK603 (YIELDGARD RW+RR), MON87403, MON87411, MON87419, MON87427 (ROUNDUP READY Maize), MON87427×59122, MON87427×MON88017, MON87427×MON88017×59122, MON87427×MON89034, MON87427×MON89034×59122, MON87427×MON89034×MIR162×MON87411, MON87427×MON89034×MON88017, MON87427×MON89034×MON88017×59122, MON87427×MON89034×NK603, MON87427×MON89034×TC1507, MON87427×MON89034×TC1507×59122, MON87427×MON89034×TC1507×MON87411×59122, MON87427×MON89034×TC1507×MON87411×59122×DAS40278, MON87427×MON89034×TC1507×MON88017, MON87427×MON89034×MIR162×NK603, MON87427×MON89034×TC1507×MON88017×59122, MON87427×TC1507, MON87427×TC1507×59122, MON87427×TC1507×MON88017, MON87427×TC1507×MON88017×59122, MON87460 (GENUITY DROUGHTGARD), MON87460×MON88017, MON87460×MON89034×MON88017, MON87460×MON89034×NK603, MON87460×NK603, MON88017, MON88017×DAS40278, MON89034, MON89034×59122, MON89034×59122×DAS40278, MON89034×59122×MON88017, MON89034×59122×MON88017×DAS40278, MON89034×DAS40278, MON89034×MON87460, MON89034×MON88017 (GENUITY VT Triple Pro), MON89034×MON88017×DAS40278, MON89034×NK603 (GENUITY VT Double Pro), MON89034×NK603×DAS40278, MON89034×TC1507, MON89034×TC1507×59122, MON89034×TC1507×59122×DAS40278, MON89034×TC1507×DAS40278, MON89034×TC1507×MON88017, MON89034×TC1507×MON88017×59122 (GENUITY SMARTSTAX), MON89034×TC1507×MON88017×59122×DAS40278, MON89034×TC1507×MON88017×DAS40278, MON89034×TC1507×NK603 (POWER CORE), MON89034×TC1507×NK603×DAS40278, MON89034×TC1507×NK603×MIR162, MON89034×TC1507×NK603×MIR162×DAS40278, MON89034×GA21, MS3 (INVIGOR Maize), MS6 (INVIGOR Maize), MZHG0JG, MZIR098, NK603 (ROUNDUP READY 2 Maize), NK603×MON810×4114×MIR604, NK603×MON810 (YIELDGARD CB+RR), NK603×T25 (ROUNDUP READY LIBERTY LINK Maize), T14 (LIBERTY LINK Maize), T25 (LIBERTY LINK Maize), T25×MON810 (LIBERTY LINK YIELDGARD Maize), TC1507 (HERCULEX I, HERCULEX CB), TC1507×59122×MON810×MIR604×NK603 (OPTIMUM INTRASECT XTREME), TC1507×MON810×MIR604×NK603, TC1507×5307, TC1507×5307×GA21, TC1507×59122 (HERCULEX XTRA), TC1507×59122×DAS40278, TC1507×59122×MON810, TC1507×59122×MON810×MIR604, TC1507×59122×MON810×NK603 (OPTIMUM INTRASECT XTRA), TC1507×59122×MON88017, TC1507×59122×MON88017×DAS40278, TC1507×59122×NK603 (HERCULEX XTRA RR), TC1507×59122×NK603×MIR604, TC1507×DAS40278, TC1507×GA21, TC1507×MIR162×NK603, TC1507×MIR604×NK603 (OPTIMUM TRISECT), TC1507×MON810, TC1507×MON810×MIR162, TC1507×MON810×MIR162×NK603, TC1507×MON810×MIR604, TC1507×MON810×NK603 (OPTIMUM INTRASECT), TC1507×MON810×NK603×MIR604, TC1507×MON88017, TC1507×MON88017×DAS40278, TC1507×NK603 (HERCULEX I RR), TC1507×NK603×DAS40278, TC6275, and VCO-01981-5.

Additional Genetically Modified Plants

The methods and bacteria described herein are suitable for any of a variety of genetically modified plants or part thereof.

Furthermore, the methods and bacteria described herein are suitable for any of the following genetically modified plant events (see Tables 14-19) which have been approved in one or more countries.

TABLE 14 Rice Traits, which can be combined with microbes of the disclosure Oryza sativa Rice Event Company Description CL121, CL141, CFX51 BASF Inc. Tolerance to the imidazolinone herbicide, imazethapyr, induced by chemical mutagenesis of the acetolactate synthase (ALS) enzyme using ethyl methanesulfonate (EMS). IMINTA-1, IMINTA-4 BASF Inc. Tolerance to imidazolinone herbicides induced by chemical mutagenesis of the acetolactate synthase (ALS) enzyme using sodium azide. LLRICE06, LLRICE62 Aventis CropScience Glufosinate ammonium herbicide tolerant rice produced by inserting a modified phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces hygroscopicus). LLRICE601 Bayer CropScience (Aventis Glufosinate ammonium herbicide CropScience(AgrEvo)) tolerant rice produced by inserting a modified phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces hygroscopicus). PWC16 BASF Inc. Tolerance to the imidazolinone herbicide, imazethapyr, induced by chemical mutagenesis of the acetolactate synthase (ALS) enzyme using ethyl methanesulfonate (EMS).

TABLE 15 Alfalfa Traits, which can be combined with microbes of the disclosure Medicago sativa Alfalfa Event Company Description J101, J163 Monsanto Company and Glyphosate herbicide tolerant Forage Genetics alfalfa (lucerne) produced by International inserting a gene encoding the enzyme 5-enolypyruvylshikimate- 3-phosphate synthase (EPSPS) from the CP4 strain of Agrobacterium tumefaciens.

TABLE 16 Wheat Traits, which can be combined with microbes of the disclosure Triticum aestivum Wheat Event Company Description AP205CL BASF Inc. Selection for a mutagenized version of the enzyme acetohydroxyacid synthase (AHAS), also known as acetolactate synthase (ALS) or acetolactate pyruvate-lyase. AP602CL BASF Inc. Selection for a mutagenized version of the enzyme acetohydroxyacid synthase (AHAS), also known as acetolactate synthase (ALS) or acetolactate pyruvate-lyase. BW255-2, BW238-3 BASF Inc. Selection for a mutagenized version of the enzyme acetohydroxyacid synthase (AHAS), also known as acetolactate synthase (ALS) or acetolactate pyruvate-lyase. BW7 BASF Inc. Tolerance to imidazolinone herbicides induced by chemical mutagenesis of the acetohydroxyacid synthase (AHAS) gene using sodium azide. MON71800 Monsanto Company Glyphosate tolerant wheat variety produced by inserting a modified 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from the soil bacterium Agrobacterium tumefaciens, strain CP4. SWP965001 Cyanamid Crop Selection for a mutagenized version Protection of the enzyme acetohydroxyacid synthase (AHAS), also known as acetolactate synthase (ALS) or acetolactate pyruvate-lyase. Teal 11A BASF Inc. Selection for a mutagenized version of the enzyme acetohydroxyacid synthase (AHAS), also known as acetolactate synthase (ALS) or acetolactate pyruvate-lyase.

TABLE 17 Sunflower Traits, which can be combined with microbes of the disclosure Helianthus annuus Sunflower Event Company Description X81359 BASF Inc. Tolerance to imidazolinone herbicides by selection of a naturally occurring mutant.

TABLE 18 Soybean Traits, which can be combined with microbes of the disclosure Glycine max L. Soybean Event Company Description A2704-12, A2704-21, Bayer CropScience Glufosinate ammonium herbicide A5547-35 (Aventis CropScience tolerant soybean produced by (AgrEvo)) inserting a modified phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces viridochromogenes. A5547-127 Bayer CropScience Glufosinate ammonium herbicide (Aventis CropScience tolerant soybean produced by (AgrEvo)) inserting a modified phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces viridochromogenes. BPS-CV127-9 BASF Inc. The introduced csr1-2 gene from Arabidopsis thaliana encodes an acetohydroxyacid synthase protein that confers tolerance to imidazolinone herbicides due to a point mutation that results in a single amino acid substitution in which the serine residue at position 653 is replaced by asparagine (S653N). DP-3 05423 Pioneer Hi-Bred High oleic acid soybean produced International Inc. by inserting additional copies of a portion of the omega 6 desaturase encoding gene, gm-fad2-1 resulting in silencing of the endogenous omega-6 desaturase gene (FAD2-1). DP356043 Pioneer Hi-Bred Soybean event with two herbicide International Inc. tolerance genes: glyphosate N- acetlytransferase, which detoxifies glyphosate, and a modified acetolactate synthase (ALS) gene which is tolerant to ALS-inhibiting herbicides. G94-1, G94-19, G168 DuPont Canada High oleic acid soybean produced Agricultural Products by inserting a second copy of the fatty acid desaturase (Gm Fad2-1) encoding gene from soybean, which resulted in “silencing” of the endogenous host gene. GTS 40-3-2 Monsanto Company Glyphosate tolerant soybean variety produced by inserting a modified 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from the soil bacterium Agrobacterium tumefaciens. GU262 Bayer CropScience Glufosinate ammonium herbicide (Aventis tolerant soybean produced by CropScience(AgrEvo)) inserting a modified phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces viridochromogenes. MON87701 Monsanto Company Resistance to Lepidopteran pests of soybean including velvetbean caterpillar (Anticarsia gemmatalis) and soybean looper (Pseudoplusia includens). MON87701 × Monsanto Company Glyphosate herbicide tolerance MON89788 through expression of the EPSPS encoding gene from A. tumefaciens strain CP4, and resistance to Lepidopteran pests of soybean including velvetbean caterpillar (Anticarsia gemmatalis) and soybean looper (Pseudoplusia includens) via expression of the Cry1Ac encoding gene from B. thuringiensis. MON89788 Monsanto Company Glyphosate-tolerant soybean produced by inserting a modified 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding aroA (epsps) gene from Agrobacterium tumefaciens CP4. OT96-15 Agriculture & Agri-Food Low linolenic acid soybean Canada produced through traditional cross- breeding to incorporate the novel trait from a naturally occurring fan1 gene mutant that was selected for low linolenic acid. W62, W98 Bayer CropScience Glufosinate ammonium herbicide (Aventis tolerant soybean produced by CropScience(AgrEvo)) inserting a modified phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces hygroscopicus.

TABLE 19 Corn Traits, which can be combined with microbes of the disclosure Zea mays L. Maize Event Company Description 176 Syngenta Seeds, Inc. Insect-resistant maize produced by inserting the Cry1Ab gene from Bacillus thuringiensis subsp. kurstaki. The genetic modification affords resistance to attack by the European corn borer (ECB). 3751 IR Pioneer Hi-Bred Selection of somaclonal variants 676, 678, 680 International Inc. by culture of embryos on Pioneer Hi-Bred imidazolinone containing media. International Inc. Male-sterile and glufosinate ammonium herbicide tolerant maize produced by inserting genes encoding DNA adenine methylase and phosphinothricin acetyltransferase (PAT) from Escherichia coli and Streptomyces viridochromogenes, respectively. B16 (DLL25) Dekalb Genetics Glufosinate ammonium herbicide Corporation tolerant maize produced by inserting the gene encoding phosphinothricin acetyltransferase (PAT) from Streptomyces hygroscopicus. BT11 (X4334CBR, Syngenta Seeds, Inc. Insect-resistant and herbicide X4734CBR) tolerant maize produced by inserting the Cry1Ab gene from Bacillus thuringiensis subsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT) encoding gene from S. viridochromogenes. BT11 × GA21 Syngenta Seeds, Inc. Stacked insect resistant and herbicide tolerant maize produced by conventional cross breeding of parental lines BT11 (OECD unique identifier: SYN-BTO11-1) and GA21 (OECD unique identifier: MON-OOO21-9). BT11 × MIR162 × Syngenta Seeds, Inc. Resistance to Coleopteran pests, MIR604 × GA21 particularly corn rootworm pests (Diabrotica spp.) and several Lepidopteran pests of corn, including European corn borer (ECB, Ostrinia nubilalis), corn earworm (CEW, Helicoverpa zea), fall army worm (FAW, Spodoptera frugiperda), and black cutworm (BCW, Agrotis ipsilon); tolerance to glyphosate and glufosinate- ammonium containing herbicides. BT11 × MIR162 Syngenta Seeds, Inc. Stacked insect resistant and herbicide tolerant maize produced by conventional cross breeding of parental lines BT11 (OECD unique identifier: SYN-BTO11-1) and MIR162 (OECD unique identifier: SYN-1R162-4). Resistance to the European Corn Borer and tolerance to the herbicide glufosinate ammonium (Liberty) is derived from BT11, which contains the Cry1Ab gene from Bacillus thuringiensis subsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT) encoding gene from S. viridochromogenes. Resistance to other Lepidopteran pests, including H. zea, S. frugiperda, A. ipsilon, and S. albicosta, is derived from MIR162, which contains the vip3Aa gene from Bacillus thuringiensis strain AB88. BT11 × MIR162 × Syngenta Seeds, Inc. Bacillus thuringiensis Cry1Ab MIR604 delta-endotoxin protein and the genetic material necessary for its production (via elements of vector pZO1502) in Event Bt11 corn (OECD Unique Identifier: SYNBTO11-1) × Bacillus thuringiensis Vip3Aa20 insecticidal protein and the genetic material necessary for its production (via elements of vector pNOV1300) in Event MIR162 maize (OECD Unique Identifier: SYN-IR162-4) × modified Cry3A protein and the genetic material necessary for its production (via elements of vector pZM26) in Event MIR604 corn (OECD Unique Identifier: SYN-1R604-5). CBH-351 Aventis CropScience Insect-resistant and glufosinate ammonium herbicide tolerant maize developed by inserting genes encoding Cry9C protein from Bacillus thuringiensis subsp tolworthi and phosphinothricin acetyltransferase (PAT) from Streptomyces hygroscopicus. DAS-06275-8 DOW AgroSciences LLC Lepidopteran insect resistant and glufosinate ammonium herbicide- tolerant maize variety produced by inserting the Cry1F gene from Bacillus thuringiensis var aizawai and the phosphinothricin acetyltransferase (PAT) from Streptomyces hygroscopicus. BT11 × MIR604 Syngenta Seeds, Inc. Stacked insect resistant and herbicide tolerant maize produced by conventional cross breeding of parental lines BT11 (OECD unique identifier: SYN-BTO11-1) and MIR604 (OECD unique identifier: SYN-1R6O5-5). Resistance to the European Corn Borer and tolerance to the herbicide glufosinate ammonium (Liberty) is derived from BT11, which contains the Cry1Ab gene from Bacillus thuringiensis subsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT) encoding gene from S. viridochromogenes. Corn rootworm -resistance is derived from MIR604 which contains the mCry3A gene from Bacillus thuringiensis. BT11 × MIR604 × Syngenta Seeds, Inc. Stacked insect resistant and GA21 herbicide tolerant maize produced by conventional cross breeding of parental lines BT11 (OECD unique identifier: SYN-BTO11-1), MIR604 (OECD unique identifier: SYN-1R6O5-5) and GA21 (OECD unique identifier: MON- OOO21-9). Resistance to the European Corn Borer and tolerance to the herbicide glufosinate ammonium (Liberty) is derived from BT11, which contains the Cry1Ab gene from Bacillus thuringiensis subsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT) encoding gene from S. viridochromogenes. Corn rootworm-resistance is derived from MIR604 which contains the mCry3A gene from Bacillus thuringiensis. Tolerance to glyphosate herbicide is derived from GA21 which contains a a modified EPSPS gene from maize. DAS-59122-7 DOW AgroSciences LLC Corn rootworm-resistant maize and Pioneer Hi-Bred produced by inserting the International Inc. Cry34Ab1 and Cry35Ab1 genes from Bacillus thuringiensis strain PS149B1. The PAT encoding gene from Streptomyces viridochromogenes was introduced as a selectable marker. DAS-59122-7 × TC1507 × DOW AgroSciences LLC Stacked insect resistant and NK603 and Pioneer Hi-Bred herbicide tolerant maize produced International Inc. by conventional cross breeding of parental lines DAS-59122-7 (OECD unique identifier: DAS- 59122-7) and TC1507 (OECD unique identifier: DAS-01507-1) with NK603 (OECD unique identifier: MON-00603-6). Corn rootworm-resistance is derived from DAS-59122- 7 which contains the Cry34Ab1 and Cry35Ab1 genes from Bacillus thuringiensis strain P5149B1. Lepidopteran resistance and tolerance to glufosinate ammonium herbicide is derived from TC1507. Tolerance to glyphosate herbicide is derived from NK603. DBT418 Dekalb Genetics Insect-resistant and glufosinate Corporation ammonium herbicide tolerant maize developed by inserting genes encoding Cry1AC protein from Bacillus thuringiensis subsp kurstaki and phosphinothricin acetyltransferase (PAT) from Streptomyces hygroscopicus. MIR604 × GA21 Syngenta Seeds, Inc. Stacked insect resistant and herbicide tolerant maize produced by conventional cross breeding of parental lines MIR604 (OECD unique identifier: SYN-1R605-5) and GA21 (OECD unique identifier: MON-00021-9). Corn rootworm-resistance is derived from MIR604 which contains the mCry3A gene from Bacillus thuringiensis. Tolerance to glyphosate herbicide is derived from GA21. MON80100 Monsanto Company Insect-resistant maize produced by inserting the Cry1Ab gene from Bacillus thuringiensis subsp. kurstaki. The genetic modification affords resistance to attack by the European corn borer (ECB). MON802 Monsanto Company Insect-resistant and glyphosate herbicide tolerant maize produced by inserting the genes encoding the Cry1Ab protein from Bacillus thuringiensis and the 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS) from A. tumefaciens strain CP4. MON809 Pioneer Hi-Bred Resistance to European corn borer International Inc. (Ostrinia nubilalis) by introduction of a synthetic Cry1Ab gene. Glyphosate resistance via introduction of the bacterial version of a plant enzyme, 5-enolpynivyl shikimate-3- phosphate synthase (EPSPS). MON810 Monsanto Company Insect-resistant maize produced by inserting a truncated form of the Cry1Ab gene from Bacillus thuringiensis subsp. kurstaki HD-1. The genetic modification affords resistance to attack by the European corn borer (ECB). MONS10 × LY038 Monsanto Company Stacked insect resistant and enhanced lysine content maize derived from conventional crossbreeding of the parental lines MONS10 (OECD identifier: MON-OO81O-6) and LY038 (OECD identifier: REN-OOO38- 3). MON810 × MON88017 Monsanto Company Stacked insect resistant and glyphosate tolerant maize derived from conventional cross-breeding of the parental lines MON810 (OECD identifier: MON-OO81O- 6) and MON88017 (OECD identifier: MON-88017-3). European corn borer (ECB) resistance is derived from a truncated form of the Cry1Ab gene from Bacillus thuringiensis subsp. kurstaki HD-1 present in MON810. Corn rootworm resistance is derived from the Cry3Bb1 gene from Bacillus thuringiensis subspecies kumamotoensis strain EG4691 present in MON88017. Glyphosate tolerance is derived from a 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from Agrobacterium tumefaciens strain CP4 present in MON88017. MON832 Monsanto Company Introduction, by particle bombardment, of glyphosate oxidase (GOX) and a modified 5- enolpyruvyl shikimate-3-phosphate synthase (EPSPS), an enzyme involved in the shikimate biochemical pathway for the production of the aromatic amino acids. MON863 Monsanto Company Corn rootworm resistant maize produced by inserting the Cry3Bb1 gene from Bacillus thuringiensis subsp. kumamotoensis. MON863 × MONS10 Monsanto Company Stacked insect resistant corn hybrid derived from conventional cross-breeding of the parental lines MON863 (OECD identifier: MON-00863-5) and MON810 (OECD identifier: MON-00810-6) MON863 × MONS10 × Monsanto Company Stacked insect resistant and Monsanto NK603 herbicide tolerant corn hybrid derived from conventional crossbreeding of the stacked hybrid MON-00863-5 × MON- 00810-6 and NK603 (OECD identifier: MON-00603-6). MON863 × NK603 Monsanto Company Stacked insect resistant and herbicide tolerant corn hybrid derived from conventional crossbreeding of the parental lines MON863 (OECD identifier: MON-OO863-5) and NK603 (OECD identifier: MON-OO6O3- 6). MON87460 Monsanto Company MON 87460 was developed to provide reduced yield loss under water-limited conditions compared to conventional maize. Efficacy in MON 87460 is derived by expression of the inserted Bacillus subtilis cold shock protein B (CspB). MON88017 Monsanto Company Corn rootworm-resistant maize produced by inserting the Cry3Bb1 gene from Bacillus thuringiensis subspecies kumamotoensis strain EG4691. Glyphosate tolerance derived by inserting a 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from Agrobacierium tumefaciens strain CP4. MON89034 Monsanto Company Maize event expressing two different insecticidal proteins from Bacillus thuringiensis providing resistance to number of Lepidopteran pests. MON89034 × Monsanto Company Stacked insect resistant and MON88017 glyphosate tolerant maize derived from conventional cross-breeding of the parental lines MON89034 (OECD identifier: MON-89O34-3) and MON88017 (OECD identifier: MON-88O17-3). Resistance to Lepidopteran insects is derived from two Cry genes present in MON89043. Corn rootworm resistance is derived from a single Cry genes and glyphosate tolerance is derived from the 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encoding gene from Agrobacterium tumefaciens present in MON88017. MON89034 × NK603 Monsanto Company Stacked insect resistant and herbicide tolerant maize produced by conventional cross breeding of parental lines MON89034 (OECD identifier: MON-89034-3) with NK603 (OECD unique identifier: MON-00603-6). Resistance to Lepidopteran insects is derived from two Cry genes present in MON89043. Tolerance to glyphosate herbicide is derived from NK603. NK603 × MON810 Monsanto Company Stacked insect resistant and herbicide tolerant corn hybrid derived from conventional crossbreeding of the parental lines NK603 (OECD identifier: MON- 00603-6) and MONS10 (OECD identifier: MON-00810-6). MON89034 × TC1507 × Monsanto Company and Stacked insect resistant and MON88017 × DAS- Mycogen Seeds c/o Dow herbicide tolerant maize produced 59122-7 AgroSciences LLC by conventional cross breeding of parental lines: MON89034, TC1507, MON88017, and DAS-59 122. Resistance to the above- ground and below-ground insect pests and tolerance to glyphosate and glufosinate-ammonium containing herbicides. M53 Bayer CropScience Male sterility caused by expression (Aventis of the barnase ribonuclease gene CropScience(AgrEvo)) from Bacillus amyloliquefaciens; PPT resistance was via PPT- acetyltransferase (PAT). M56 Bayer CropScience Male sterility caused by expression (Aventis of the barnase ribonuclease gene CropScience(AgrEvo) from Bacillus amyloliquefaciens; PPT resistance was via PPT- acetyltransferase (PAT). NK603 Monsanto Company Introduction, by particle bombardment, of a modified 5- enolpyruvyl shikimate-3-phosphate synthase (EPSPS), an enzyme involved in the shikimate biochemical pathway for the production of the aromatic amino acids. NK603 × T25 Monsanto Company Stacked glufosinate ammonium and glyphosate herbicide tolerant maize hybrid derived from conventional cross-breeding of the parental lines NK603 (OECD identifier: MON-00603-6) and T25 (OECD identifier: ACS-ZM003- 2). T25 × MON810 Bayer CropScience Stacked insect resistant and (Aventis herbicide tolerant corn hybrid CropScience(AgrEvo)) derived from conventional crossbreeding of the parental lines T25 (OECD identifier: ACS- ZMOO3-2) and MON810 (OECD identifier: MON-OO81O-6). TC1507 Mycogen (c/o Dow Insect-resistant and glufosinate AgroSciences); Pioneer ammonium herbicide tolerant (c/o DuPont) maize produced by inserting the Cry1F gene from Bacillus thuringiensis var. aizawai and the phosphinothricin N-acetyltransferase encoding gene from Streptomyces viridochromogenes. TC1507 × NK603 DOW AgroSciences LLC Stacked insect resistant and herbicide tolerant corn hybrid derived from conventional crossbreeding of the parental lines 1507 (OECD identifier: DAS- O1507-1) and NK603 (OECD identifier: MON-OO6O3-6). TC1507 × DAS-59122-7 DOW AgroSciences LLC Stacked insect resistant and and Pioneer Hi-Bred herbicide tolerant maize produced International Inc. by conventional cross breeding of parental lines TC1507 (OECD unique identifier: DAS-O15O7-1) with DAS-59122-7 (OECD unique identifier: DAS-59122-7). Resistance to Lepidopteran insects is derived from TC1507 due the presence of the Cry1F gene from Bacillus thuringiensis var. aizawai. Corn rootworm-resistance is derived from DAS-59122-7 which contains the Cry34Ab1 and Cry35Ab1 genes from Bacillus thuringiensis strain P5149B1. Tolerance to glufosinate ammonium herbicide is derived from TC1507 from the phosphinothricin N-acetyltransferase encoding gene from Streptomyces viridochromogenes. Event Company Description Hybrid Family P0157 Dupont Pioneer P0157 P0157AM Dupont Pioneer AM LL RR2 P0157 P0157AMXT Dupont Pioneer AMXT LL RR2 P0157 P0157R Dupont Pioneer RR2 P0157 P0339AM Dupont Pioneer AM LL RR2 P0339 P0339AMXT Dupont Pioneer AMXT LL RR2 P0339 P0306AM Dupont Pioneer AM LL RR2 P0306 P0589 Dupont Pioneer P0589 P0589AM Dupont Pioneer AM LL RR2 P0589 P0589AMXT Dupont Pioneer AMXT LL RR2 P0589 P0589R Dupont Pioneer RR2 P0589 P0574 Dupont Pioneer P0574 P0574AM Dupont Pioneer AM LL RR2 P0574 P0574AMXT Dupont Pioneer AMXT LL RR2 P0574 P0533EXR Dupont Pioneer HXX LL RR2 P0533 P0506AM Dupont Pioneer AM LL RR2 P0566 P0760AMXT Dupont Pioneer AMXT LL RR2 P0760 P0707AM Dupont Pioneer AM LL RR2 P0707 P0707AMXT Dupont Pioneer AMXT LL RR2 P0707 P0825AM Dupont Pioneer AM LL RR2 P0825 P0825AMXT Dupont Pioneer AMXT LL RR2 P0825 P0969AM Dupont Pioneer AM LL RR2 P0969 P0969AMXT Dupont Pioneer AMXT LL RR2 P0969 P0937AM Dupont Pioneer AM LL RR2 P0937 P0919AM Dupont Pioneer AM LL RR2 P0919 P0905EXR Dupont Pioneer HXX LL RR2 P0905 P1197 Dupont Pioneer P1197 P1197AM Dupont Pioneer AM LL RR2 P1197 P1197AMXT Dupont Pioneer AMXT LL RR2 P1197 P1197R Dupont Pioneer RR2 P1197 P1151 Dupont Pioneer P1151 P1151AM Dupont Pioneer AM LL RR2 P1151 P1151R Dupont Pioneer RR2 P1151 P1138AM Dupont Pioneer AM LL RR2 P1138 P1366AM Dupont Pioneer AM LL RR2 P1366 P1366AMXT Dupont Pioneer AMXT LL RR2 P1366 P1365AMX Dupont Pioneer AMX LL RR2 P1365 P1353AM Dupont Pioneer AM LL RR2 P1353 P1345 Dupont Pioneer P1345 P1311AMXT Dupont Pioneer AMXT LL RR2 P1311 P1498EHR Dupont Pioneer HX1 LL RR2 P1498 P1498R Dupont Pioneer RR2 P1498 P1443AM Dupont Pioneer AM LL RR2 P1443 P1555CHR Dupont Pioneer RW HX1 LL RR2 P1555 P1751AMT Dupont Pioneer AMT LL RR2 P1751 P2089AM Dupont Pioneer AM LL RR2 P2089 QROME Dupont Pioneer Q LL RR2

The following are the definitions for the shorthand occurring in Table 19. AM—OPTIMUM ACREMAX Insect Protection system with YGCB, HX1, LL, RR2. AMT—OPTIMUM ACREMAX TRISECT Insect Protection System with RW,YGCB,HX1,LL,RR2. AMXT—(OPTIMUM ACREMAX XTreme). HXX—HERCULEX XTRA contains the Herculex I and Herculex RW genes. HX1—Contains the HERCULEX I Insect Protection gene which provides protection against European corn borer, southwestern corn borer, black cutworm, fall armyworm, western bean cutworm, lesser corn stalk borer, southern corn stalk borer, and sugarcane borer; and suppresses corn earworm. LL—Contains the LIBERTYLINK gene for resistance to LIBERTY herbicide. RR2—Contains the ROUNDUP READY Corn 2 trait that provides crop safety for over-the-top applications of labeled glyphosate herbicides when applied according to label directions. YGCB—contains the YTELDGARD Corn Borer gene offers a high level of resistance to European corn borer, southwestern corn borer, and southern cornstalk borer; moderate resistance to corn earworm and common stalk borer; and above average resistance to fall armyworm. RW—contains the AGRISURE root worm resistance trait. Q—provides protection or suppression against susceptible European corn borer, southwestern corn borer, black cutworm, fall armyworm, lesser corn stalk borer, southern corn stalk borer, stalk borer, sugarcane borer, and corn earworm; and also provides protection from larval injury caused by susceptible western corn rootworm, northern corn rootworm, and Mexican corn root worm; contains (1) HERCULEX XTRA Insect Protection genes that produce Cry1F and Cry34ab1 and Cry35ab1 proteins, (2) AGRISURE RW trait that includes a gene that produces mCry3A protein, and (3) YIELDGARD Corn Borer gene which produces Cry1Ab protein.

Concentrations and Rates of Application of Agricultural Compositions

As aforementioned, the agricultural compositions of the present disclosure, which comprise a taught microbe, can be applied to plants in a multitude of ways. In two particular aspects, the disclosure contemplates an in-furrow treatment or a seed treatment.

For seed treatment embodiments, the microbes of the disclosure can be present on the seed in a variety of concentrations. For example, the microbes can be found in a seed treatment at a cfu concentration, per seed of: 1×10¹, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, or more. In particular aspects, the seed treatment compositions comprise about 1×10⁴ to about 1×10⁸ cfu per seed. In other particular aspects, the seed treatment compositions comprise about 1×10⁵ to about 1×10⁷ cfu per seed. In other aspects, the seed treatment compositions comprise about 1×10⁶ cfu per seed.

In the United States, about 10% of corn acreage is planted at a seed density of above about 36.000 seeds per acre; ⅓ of the corn acreage is planted at a seed density of between about 33,000 to 36,000 seeds per acre; ⅓ of the corn acreage is planted at a seed density of between about 30.000 to 33,000 seeds per acre, and the remainder of the acreage is variable. See, “Corn Seeding Rate Considerations,” written by Steve Butzen, available at: www.pioneer.com/home/site/us/agronomy/library/corn-seeding-rate-considerations/

Table 20 below utilizes various cfu concentrations per seed in a contemplated seed treatment embodiment (rows across) and various seed acreage planting densities (1^(st) column: 15K-41K) to calculate the total amount of cfu per acre, which would be utilized in various agricultural scenarios (i.e. seed treatment concentration per seed×seed density planted per acre). Thus, if one were to utilize a seed treatment with 1×10⁶ cfu per seed and plant 30,000 seeds per acre, then the total cfu content per acre would be 3×10¹⁰ (i.e. 30K×1×10⁶).

TABLE 20 Total CFU Per Acre Calculation for Seed Treatment Embodiments Corn Population (i.e. seeds per acre) 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 15,000 1.50E+06 1.50E+07 1.50E+08 1.50E+09 1.50E+10 1.50E+11 1.50E+12 1.50E+13 16,000 1.60E+06 1.60E+07 1.60E+08 1.60E+09 1.60E+10 1.60E+11 1.60E+12 1.60E+13 17,000 1.70E+06 1.70E+07 1.70E+08 1.70E+09 1.70E+10 1.70E+11 1.70E+12 1.70E+13 18,000 1.80E+06 1.80E+07 1.80E+08 1.80E+09 1.80E+10 1.80E+11 1.80E+12 1.80E+13 19,000 1.90E+06 1.90E+07 1.90E+08 1.90E+09 1.90E+10 1.90E+11 1.90E+12 1.90E+13 20,000 2.00E+06 2.00E+07 2.00E+08 2.00E+09 2.00E+10 2.00E+11 2.00E+12 2.00E+13 21,000 2.10E+06 2.10E+07 2.10E+08 2.10E+09 2.10E+10 2.10E+11 2.10E+12 2.10E+13 22,000 2.20E+06 2.20E+07 2.20E+08 2.20E+09 2.20E+10 2.20E+11 2.20E+12 2.20E+13 23,000 2.30E+06 2.30E+07 2.30E+08 2.30E+09 2.30E+10 2.30E+11 2.30E+12 2.30E+13 24,000 2.40E+06 2.40E+07 2.40E+08 2.40E+09 2.40E+10 2.40E+11 2.40E+12 2.40E+13 25,000 2.50E+06 2.50E+07 2.50E+08 2.50E+09 2.50E+10 2.50E+11 2.50E+12 2.50E+13 26,000 2.60E+06 2.60E+07 2.60E+08 2.60E+09 2.60E+10 2.60E+11 2.60E+12 2.60E+13 27,000 2.70E+06 2.70E+07 2.70E+08 2.70E+09 2.70E+10 2.70E+11 2.70E+12 2.70E+13 28,000 2.80E+06 2.80E+07 2.80E+08 2.80E+09 2.80E+10 2.80E+11 2.80E+12 2.80E+13 29,000 2.90E+06 2.90E+07 2.90E+08 2.90E+09 2.90E+10 2.90E+11 2.90E+12 2.90E+13 30,000 3.00E+06 3.00E+07 3.00E+08 3.00E+09 3.00E+10 3.00E+11 3.00E+12 3.00E+13 31,000 3.10E+06 3.10E+07 3.10E+08 3.10E+09 3.10E+10 3.10E+11 3.10E+12 3.10E+13 32,000 3.20E+06 3.20E+07 3.20E+08 3.20E+09 3.20E+10 3.20E+11 3.20E+12 3.20E+13 33,000 3.30E+06 3.30E+07 3.30E+08 3.30E+09 3.30E+10 3.30E+11 3.30E+12 3.30E+13 34,000 3.40E+06 3.40E+07 3.40E+08 3.40E+09 3.40E+10 3.40E+11 3.40E+12 3.40E+13 35,000 3.50E+06 3.50E+07 3.50E+08 3.50E+09 3.50E+10 3.50E+11 3.50E+12 3.50E+13 36,000 3.60E+06 3.60E+07 3.60E+08 3.60E+09 3.60E+10 3.60E+11 3.60E+12 3.60E+13 37,000 3.70E+06 3.70E+07 3.70E+08 3.70E+09 3.70E+10 3.70E+11 3.70E+12 3.70E+13 38,000 3.80E+06 3.80E+07 3.80E+08 3.80E+09 3.80E+10 3.80E+11 3.80E+12 3.80E+13 39,000 3.90E+06 3.90E+07 3.90E+08 3.90E+09 3.90E+10 3.90E+11 3.90E+12 3.90E+13 40,000 4.00E+06 4.00E+07 4.00E+08 4.00E+09 4.00E+10 4.00E+11 4.00E+12 4.00E+13 41,000 4.10E+06 4.10E+07 4.10E+08 4.10E+09 4.10E+10 4.10E+11 4.10E+12 4.10E+13

For in-furrow embodiments, the microbes of the disclosure can be applied at a cfu concentration per acre of: 1×10⁶, 3.20×10¹⁰, 1.60×10¹¹, 3.20×10¹¹, 8.0×10¹¹, 1.6×10¹², 3.20×10¹², or more. Therefore, in aspects, the liquid in-furrow compositions can be applied at a concentration of between about 1×10⁶ to about 3×10¹² cfu per acre.

In some aspects, the in-furrow compositions are contained in a liquid formulation. In the liquid in-furrow embodiments, the microbes can be present at a cfu concentration per milliliter of: 1×10¹, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, or more. In certain aspects, the liquid in-furrow compositions comprise microbes at a concentration of about 1×10⁶ to about 1×10¹¹ cfu per milliliter. In other aspects, the liquid in-furrow compositions comprise microbes at a concentration of about 1×10⁷ to about 1×10¹⁰ cfu per milliliter. In other aspects, the liquid in-furrow compositions comprise microbes at a concentration of about 1×10⁸ to about 1×10⁹ cfu per milliliter. In other aspects, the liquid in-furrow compositions comprise microbes at a concentration of up to about 1×10¹³ cfu per milliliter.

DNA Barcoding

As used herein, the term “barcode,” “barcodes” or “DNA barcodes” can refer to nucleic acid codes or sequences associated with a target within a sample. A barcode can be, for example, a nucleic acid label. A barcode can be an entirely or partially amplifiable barcode. A barcode can be entirely or partially sequenceable barcode. A barcode can be a portion of a native nucleic acid that is identifiable as distinct. A barcode can be a known sequence. A barcode can be a random sequence. A barcode can comprise a junction of nucleic acid sequences, for example a junction of a native and non-native sequence. As used herein, the term “barcode” can be used interchangeably with the terms, “index”, “tag,” or “label-tag.” Barcodes can convey information. For example, in various embodiments, barcodes can be used to determine an identity of a nucleic acid, a source of a nucleic acid, an identity of a cell, and/or a target.

In sequencing, DNA barcodes allow objective identification of a sequence's sample of origin using a short unique DNA sequence. Just as the unique pattern of bars in a universal product code (UPC) identifies each consumer product, a “DNA barcode” is a specially designed DNA sequence added during sample preparation that identifies the sample source for each read output by a DNA sequencer. DNA barcoding for sample identification was described for the very first of the next generation sequencing (NGS) platforms, the GS 20, in 2007 (Parameswaran P, Jalili R, Tao L, et al. A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing. Nucleic Acids Research. 2007; 35(19):e130. doi:10.1093/nar/gkm760). Since that time, barcoding strategies have been made commercially available by Illumina (San Diego, Calif.), Pacific Biosciences (Menlo Park, Calif.), and Thermo Fisher Scientific (Waltham, Mass.) sequencing platforms.

Barcoding individual samples with specific DNA tags allows many samples to be combined for sequencing, streamlining the workflow, increasing efficiency and reducing costs. Typically, for sequencing, DNA is extracted from each sample, and the unique identifying barcode for that sample is added during polymerase chain reaction (PCR) of each sample. After each sample undergoes a separate PCR/barcoding process, multiple PCR amplicons can be mixed together (multiplexed) for sequencing. Since the sample of origin for each amplicon can be tracked using the barcodes, all reads from a sample can be grouped together again post-sequencing. The target DNA sequence may be an amplicon, or targeted gene sequence, such as bacterial 16S rRNA gene, 23S rRNA gene, eukaryotic 18S rRNA, human HLA, microbial toxin producing genes, microbial pathogenicity genes, microbial plasmid genes, human immune system genes, immune system components and other variable genetic regions of plants and other organisms.

DNA barcodes can be selected to be of sufficient length to generate the desired number of barcodes with sufficient variability to account for common sequencing errors, generally ranging in size from about 2 to about 20 bases, but may be longer or shorter. Longer barcodes will permit higher sequence diversity and typically allow more samples to be combined. Typically, DNA barcodes are synthetic, artificially generated sequences. However, the methods disclosed herein utilize natural genomic sequences as barcodes.

In some aspects, cells such as bacterial cells, fungal cells, plant cells, animal cells, protozoal cells, or insect cells are barcoded.

In some aspects, the cells are transgenic. In some aspect, the cells are non-intergenic remodeled cells. In some aspects, the cells occur in a population of cells. In some aspects, the population of cells are homogeneous. In some aspects, the population of cells are homogenous.

In some aspects, the population of cells comprise two or more different species. In some aspects, the population of cells comprise, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 different species.

In some aspects, the population of cells comprise two or more different strains. In some aspects, the population of cells comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 different strains.

In some aspects, the population of cells comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 different species; and wherein the population further comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different strains of each species.

In some aspects, barcoding a cell with a native barcode sequence comprises evaluating the genomic DNA corresponding to the species of the cell of interest to search for potential nucleic acid sequences that can serve as barcodes and forward and reverse primer binding sites. In some aspects, the evaluation is expanded to all species of the genus of the cell of interest.

As described herein, “a native barcode polynucleotide cassette” is a polynucleotide sequence comprising only nucleic acid sequences that naturally occur in the same taxonomic genus as the cell for which the cassette will be introduced. For example, a cassette comprising nucleic acid sequences from a Bacillus is not a native barcode polynucleotide cassette if the cassette is introduced to a Klebsiella, assuming that the exact sequences are not naturally occurring in the Klebsiella. Furthermore, the native barcode polynucleotide cassette must comprise a barcode sufficient to distinguish different native barcode polynucleotide cassettes from one another. In some aspects, the native barcode polynucleotide cassette comprises two or more sequences that are in a different position relative to one another as compared to the position of the two or more sequences as they naturally occur in the genome of the native microorganism.

In some aspects, a native barcode polynucleotide cassette comprises a first nucleotide sequence, a second nucleotide sequence, and a third nucleotide sequence. One of the nucleotide sequences is a barcode and the remaining two nucleotide sequences are primer binding sites.

In some aspects, sequence for the barcode portion of a native barcode polynucleotide cassette can be selected in an in silico process. The in silico process can entail use of non-transitory computer based medium (e.g., custom Python script) that can identify discrete sequences from the genome of a strain of interest that when linked together would result in candidate barcodes for native barcode polynucleotide cassettes. The candidate barcodes for the native barcode polynucleotide cassettes can then be screened in order to filter out candidate barcodes that possess one or more of start codons, nucleic acid secondary structure, enzymatic restriction sites, unbalanced GC content and/or low base pair diversity. Candidate barcodes which pass some or all of these filters would then be selected as a barcode sequence for the native barcode polynucleotide cassette for use in the methods provided herein. Each candidate barcode can possess a constant region and a variable region as described herein. Each candidate barcode can be flanked by a forward and reverse primer binding site as described herein.

In some aspects, a native barcode polynucleotide cassette comprises a first nucleotide sequence, a second nucleotide sequence, a third nucleotide sequence, a fourth nucleic acid sequence, a fifth nucleic acid sequence, a sixth nucleic acid sequence, a seventh nucleic acid sequence, an eighth nucleic acid sequence, and a ninth nucleic acid sequence. In some aspects, native barcode polynucleotide cassette comprises at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen nucleic acid sequences.

In some aspects, one or more of the naturally occurring sequences may be selected from ribosomal DNA or internal transcribed spacer (ITS) DNA. In some aspects, one or more of the naturally occurring sequences may be selected from 16S rDNA or 18S rDNA.

In some aspects, one or more of the naturally occurring sequences may be selected from 8F, 27F, CCF, 357F, 515F, 533F, 16S.1100.F16, 804F, 1237F, 338R, 519R, CDR, 806R, 907R, 1100R, 1391R, 1392R, 1492R(l), and 1492R(s); particularly the primer binding sites.

In some aspects, one or more of the naturally occurring sequences occur in a different orientation relative to the orientation of the one or more sequences in the native barcode polynucleotide cassette. In some aspects, the one or more naturally occurring sequences all occur in the same orientation, in contrast to the orientation of the one or more sequences in the native barcode polynucleotide cassette. In some aspects, introduced barcode(s) and/or primer binding sites are selected to avoid introducing start and/or stop codons in either orientation. In some aspects, the introduced barcode(s) and/or primer binding sites are in the same relative orientation to one another in which they ordinarily occur in their native genome. In some aspects, the introduced barcode(s) and/or primer binding sites are in a different relative orientation to one another in which they ordinarily occur in their native genome.

In some aspects, the one or more naturally occurring sequences occur in a closer proximity to one another in the native barcode polynucleotide cassette than they do in the genome of the organism in which they naturally occur. In some aspects, the naturally occurring sequences in the genome of their native microorganism are separated from one another by at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 700, at least 1,000, or at least 10,000 nucleotides; whereas the naturally occurring sequences in the native barcode polynucleotide cassette are separated by fewer than 1, fewer than 2, fewer than 5, fewer than 10, fewer than 15, or fewer than 20 nucleotides. The measure of distance or proximity in nucleotide bases is a count of the number of nucleotides that separate the termini of the nucleotide sequences from one another. In some aspects, the measure of distance or proximity is between the closest termini of two naturally occurring sequences.

In some aspects, the native barcode polynucleotide cassette comprises more than one barcode sequence. In some aspects, the native barcode polynucleotide cassette comprises more than one barcode sequence, in which the first barcode sequence is unique to the species, strain, or variant in which the native barcode polynucleotide cassette has been inserted. The second barcode sequence may act as an indexing site that allows for the pooling of multiple samples, for which the indexing site identifies the pool of samples. The indexing site would allow for a second level of distinction in the cells that contain a particular indexing site. For example, the indexing site could be used to indicate which technician created a batch of cells, in which period of time the cells were created, which protocol was used to create the cells, which parental line the cells are from, etc.

Samples containing the indexing site can be pooled with other samples for ease of use of running a single batch of samples instead of numerous small batches for each separate experiment. The resulting sequencing output for pooled samples can be identified by the indexing site, and then further identified by the barcode, allowing for simple sorting of the data for further analysis.

In some aspects, the native barcode polynucleotide cassette comprises more than two sets of primer binding sites. In some aspects, the native barcode polynucleotide cassette comprises at least two sets, at least three sets, at least four sets, at least five sets, at least six sets, at least seven sets, at least eight sets, at least nine sets, or at least ten sets of primer binding sites. In some aspects, each set of primer binding sites are distinct from one another with regard to nucleotide length and/or nucleotide sequence. In some aspects, one set of primer binding sites is sufficient to amplify the entire barcode sequence or just a portion of the barcode sequence. In some aspects, one set of primer binding sites is sufficient to amplify subsections of the native barcode polynucleotide binding cassette.

In some aspects, the barcode comprises distinct regions. In some aspects, the barcode comprises a constant barcode region. As used herein, “constant barcode region” refers to population of cells comprising a barcode, wherein the barcode region that is conserved across the population of cells is the constant barcode region. In some aspects, the constant barcode region occurs at the 5′ end of the barcode sequence. In some aspects, the constant barcode region ends at the 3′ end of the barcode sequence. In some aspects, the constant barcode region is flanked on each end by a variable barcode region. In some aspects, the flanking variable barcode regions were a single variable barcode region that was bisected by the constant barcode region. In some aspects, the flanking variable barcode regions are two separate variable barcode regions.

In some aspects, the barcode comprises a variable barcode region. As used herein, “variable barcode region” refers to a population of cells comprising a barcode, wherein the barcode region that is not conserved across the population of cells is the variable barcode region. In some aspects, the variable barcode region occurs at the 5′ end of the barcode sequence. In some aspects, the variable barcode region ends at the 3′ end of the barcode. In some aspects, the variable region is flanked on each end by a constant barcode region. In some aspects, the flanking constant barcode regions were a single constant barcode region that was bisected by the variable barcode region. In some aspects, the flanking constant barcode regions are two separate constant barcode regions. In some aspects, the “variable” refers to differences in sequence identity. In some aspects, the “variable” refers to differences in sequence length.

In some aspects, a population of cells are derived from the same parent strain, and the population of cells are formed by a plurality of different strains. In this aspect, all of the cells derived from the same parent strain would share at least one constant barcode region, and only those cells of the population that are of the same strain would share the same variable barcode region. In some aspects, the population of cells could be mixed with another population of cells and the cells could by lysed, the primer binding sites amplified, and the resulting amplicons sequenced, and the resulting sequencing data could be appropriately distinguished given the presence of the one or more constant barcode regions and the one or more variable barcode regions.

In some aspects, any number of qPCR assays could be run on the populations of cells at different time points, and the resulting amplicons could be appropriately distinguished given the presence of the one or more constant barcode regions and the one or more variable barcode regions.

In some aspects, the constant and/or variable barcode regions are about 20, about 25, about 30, about 40, about 50, about 60, about 70 about 80, about 90, or about 100 nt long. In some aspects, the constant and/or variable barcode regions are at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70 at least about 80, at least about 90, or at least about 100 nt long.

In some aspects, the constant barcode region and the variable barcode region are different barcodes that are not immediately adjacent to one another. In some aspects, the barcodes are separated by about 5, about 10, about 15, about 20, about 25, about 30, about 50, about 75, about 100, about 150, about 200, about 300, about 400, about 500, about 1,000, about 1,500, about 2,000, or about 5,000 nt. In some aspects, the barcodes are separated by at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, at least about 1,000, at least about 1,500, at least about 2,000, or at least about 5,000 nt.

In some aspects, all of the barcodes are flanked by at least one forward primer binding site on one side and at least one reverse primer binding site on the other side. In some aspects, any one or more cells of any one or more organisms of the present disclosure may be barcoded according to the disclosure. In some aspects, any one or more microbes of the present disclosure may be barcoded according to the disclosure. In some aspects, the microbes may be selected from, but not limited to: Paraburkholderia tropica, Paraburkholderia xenovorans, Pseudomonas syringae, Pseudomonas benzenivorans, Metakosakonia massiliensis, Metakosakonia intestinii Pseudomonas nitroreductans, Pseudomonas abietaniphila, Pseudomonas baetica, Pseudomonas jessenii, Pseudomonas brassicacaerum, Pseudomonas extremorientalis, Pseudomonas simiae, Pseudomonas protagens, Pseudomonas stutzeri, Herbaspirillum robiniae, Herbaspirillum frisingense, Herbaspirillum huttiense, and Herbaspirillum seropedicae.

In some aspects, the barcode(s) and/or primer binding sites are modified by 1 or 2 or 3 or 4 point mutations for ease of use in cloning and workflow. In some aspects, the barcode(s) and/or primer binding sites are modified by PCR cloning with degenerate primers.

In some aspects, the primer binding sites of the present disclosure are universal primer binding sites. In some aspects, the primer binding sites are specific to the native barcode polynucleotide cassette. In some aspects, a population of cells comprising the native barcode polynucleotide cassette comprises the same reverse/forward primer binding sites in each of the cassettes. In some aspects, the primer binding sites have a GC content of at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, or at least 75%. In some aspects, the primer binding sites have a GC content of 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some aspects, the primer binding sites have a GC content of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75%.

In some aspects, the native barcode polynucleotide cassette is 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 325, 350, 375, 400, 425, 450, 475, or 500 nucleotides in length.

In some aspects, the native barcode polynucleotide cassette is about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, or about 500 nucleotides in length.

In some aspects, the native barcode polynucleotide cassette is fewer than 50, fewer than 55, fewer than 60, fewer than 65, fewer than 70, fewer than 75, fewer than 80, fewer than 85, fewer than 90, fewer than 95, fewer than 100, fewer than 105, fewer than 110, fewer than 115, fewer than 120, fewer than 125, fewer than 130, fewer than 135, fewer than 140, fewer than 145, fewer than 150, fewer than 160, fewer than 170, fewer than 180, fewer than 190, fewer than 200, fewer than 210, fewer than 220, fewer than 230, fewer than 240, fewer than 250, fewer than 260, fewer than 270, fewer than 280, fewer than 290, fewer than 300, fewer than 325, fewer than 350, fewer than 375, fewer than 400, fewer than 425, fewer than 450, fewer than 475, or fewer than 500 nucleotides in length.

In some aspects, the native barcode polynucleotide cassette is at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, or at least 500 nucleotides in length.

In some aspects, the native barcode polynucleotide cassette is about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, or about 500 nucleotides in length.

In some aspects, the native barcode polynucleotide cassette is between 50 to 500, 50 to 400, 50 to 300, 50 to 250, 50 to 200, 50 to 180, 50 to 160, 50 to 140, 50 to 120, 50 to 100, 50 to 80, 50 to 70, 50 to 60, 60 to 500, 60 to 400, 60 to 300, 60 to 250, 60 to 200, 60 to 180, 60 to 160, 60 to 140, 60 to 120, 60 to 100, 60 to 80, 60 to 70, 70 to 500, 70 to 400, 70 to 300, 70 to 250, 70 to 200, 70 to 180, 70 to 160, 70 to 140, 70 to 120, 70 to 100, 70 to 80, 80 to 500, 80 to 400, 80 to 300, 80 to 250, 80 to 200, 80 to 180, 80 to 160, 80 to 140, 80 to 120, 80 to 100, 100 to 500, 100 to 400, 100 to 300, 100 to 250, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 120 to 500, 120 to 400, 120 to 300, 120 to 250, 120 to 200, 120 to 180, 120 to 160, 120 to 140, 140 to 500, 140 to 400, 140 to 300, 140 to 250, 140 to 200, 140 to 180, 140 to 160, 160 to 500, 160 to 400, 160 to 300, 160 to 250, 160 to 200, 160 to 180, 180 to 500, 180 to 400, 180 to 300, 180 to 250, 180 to 200, 200 to 500, 200 to 400, 200 to 300, 200 to 250, 250 to 500, 250 to 400, 250 to 300, 300 to 500, 300 to 400, or 400 to 500 nucleotides in length.

In some aspects, the native barcode polynucleotide cassette is between about 50 to 500, about 50 to 400, about 50 to 300, about 50 to 250, about 50 to 200, about 50 to 180, about 50 to 160, about 50 to 140, about 50 to 120, about 50 to 100, about 50 to 80, about 50 to 70, about 50 to 60, about 60 to 500, about 60 to 400, about 60 to 300, about 60 to 250, about 60 to 200, about 60 to 180, about 60 to 160, about 60 to 140, about 60 to 120, about 60 to 100, about 60 to 80, about 60 to 70, about 70 to 500, about 70 to 400, about 70 to 300, about 70 to 250, about 70 to 200, about 70 to 180, about 70 to 160, about 70 to 140, about 70 to 120, about 70 to 100, about 70 to 80, about 80 to 500, about 80 to 400, about 80 to 300, about 80 to 250, about 80 to 200, about 80 to 180, about 80 to 160, about 80 to 140, about 80 to 120, about 80 to 100, about 100 to 500, about 100 to 400, about 100 to 300, about 100 to 250, about 100 to 200, about 100 to 180, about 100 to 160, about 100 to 140, about 100 to 120, about 120 to 500, about 120 to 400, about 120 to 300, about 120 to 250, about 120 to 200, about 120 to 180, about 120 to 160, about 120 to 140, about 140 to 500, about 140 to 400, about 140 to 300, about 140 to 250, about 140 to 200, about 140 to 180, about 140 to 160, about 160 to 500, about 160 to 400, about 160 to 300, about 160 to 250, about 160 to 200, about 160 to 180, about 180 to 500, about 180 to 400, about 180 to 300, about 180 to 250, about 180 to 200, about 200 to 500, about 200 to 400, about 200 to 300, about 200 to 250, about 250 to 500, about 250 to 400, about 250 to 300, about 300 to 500, about 300 to 400, or about 400 to 500 nucleotides in length.

In some aspects, the primer binding sites are 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length.

In some aspects, the primer binding sites are about 10, about 12, about 14, about 16, about 18, about 20, about 22, about 24, about 26, about 28, about 30, about 32, about 34, about 36, about 38, about 40, about 42, about 44, about 46, about 48, or about 50 nucleotides in length.

In some aspects, the primer binding sites are fewer than 10, fewer than 12, fewer than 14, fewer than 16, fewer than 18, fewer than 20, fewer than 22, fewer than 24, fewer than 26, fewer than 28, fewer than 30, fewer than 32, fewer than 34, fewer than 36, fewer than 38, fewer than 40, fewer than 42, fewer than 44, fewer than 46, fewer than 48, or fewer than 50 nucleotides in length.

In some aspects, the primer binding sites are at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30, at least 32, at least 34, at least 36, at least 38, at least 40, at least 42, at least 44, at least 46, at least 48, or at least 50 nucleotides in length.

In some aspects the primer binding sites are between 10 to 50, 10 to 44, 10 to 40, 10 to 36, 10 to 34, 10 to 30, 10 to 26, 10 to 24, 10 to 20, 10 to 16, 10 to 14, 14 to 50, 14 to 44, 14 to 40, 14 to 36, 14 to 34, 14 to 30, 14 to 26, 14 to 24, 14 to 20, 14 to 16, 16 to 50, 16 to 44, 16 to 40, 16 to 36, 16 to 34, 16 to 30, 16 to 26, 16 to 24, 16 to 20, 20 to 50, 20 to 44, 20 to 40, 20 to 36, 20 to 34, 20 to 30, 20 to 26, 20 to 24, 24 to 50, 24 to 44, 24 to 40, 24 to 36, 24 to 34, 24 to 30, 24 to 26, 26 to 50, 26 to 44, 26 to 40, 26 to 36, 26 to 34, 26 to 30, 30 to 50, 30 to 44, 30 to 40, 30 to 36, 30 to 34, 34 to 50, 34 to 44, 34 to 40, 34 to 36, 36 to 50, 36 to 44, 36 to 40, 40 to 50, 40 to 44, or 44 to 50 nucleotides in length.

In some aspects the primer binding sites are between about 10 to 50, about 10 to 44, about 10 to 40, about 10 to 36, about 10 to 34, about 10 to 30, about 10 to 26, about 10 to 24, about 10 to 20, about 10 to 16, about 10 to 14, about 14 to 50, about 14 to 44, about 14 to 40, about 14 to 36, about 14 to 34, about 14 to 30, about 14 to 26, about 14 to 24, about 14 to 20, about 14 to 16, about 16 to 50, about 16 to 44, about 16 to 40, about 16 to 36, about 16 to 34, about 16 to 30, about 16 to 26, about 16 to 24, about 16 to 20, about 20 to 50, about 20 to 44, about 20 to 40, about 20 to 36, about 20 to 34, about 20 to 30, about 20 to 26, about 20 to 24, about 24 to 50, about 24 to 44, about 24 to 40, about 24 to 36, about 24 to 34, about 24 to 30, about 24 to 26, about 26 to 50, about 26 to 44, about 26 to 40, about 26 to 36, about 26 to 34, about 26 to 30, about 30 to 50, about 30 to 44, about 30 to 40, about 30 to 36, about 30 to 34, about 34 to 50, about 34 to 44, about 34 to 40, about 34 to 36, about 36 to 50, about 36 to 44, about 36 to 40, about 40 to 50, about 40 to 44, or about 44 to 50 nucleotides in length.

In some aspects, the barcode is 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides in length.

In some aspects, the barcode is about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 nucleotides in length.

In some aspects, the barcode is fewer than 20, fewer than 25, fewer than 30, fewer than 35, fewer than 40, fewer than 45, fewer than 50, fewer than 55, fewer than 60, fewer than 65, fewer than 70, fewer than 75, fewer than 80, fewer than 85, fewer than 90, fewer than 95, fewer than 100, fewer than 110, fewer than 120, fewer than 130, fewer than 140, fewer than 150, fewer than 160, fewer than 170, fewer than 180, fewer than 190, or fewer than 200 nucleotides in length.

In some aspects, the barcode is at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length.

In some aspects, the barcode is at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, or at least about 200 nucleotides in length.

In some aspects, the barcode is between 20 to 200, 20 to 160, 20 to 140, 20 to 120, 20 to 100, 20 to 80, 20 to 70, 20 to 60, 20 to 40, 20 to 30, 30 to 200, 30 to 160, 30 to 140, 30 to 120, 30 to 100, 30 to 80, 30 to 70, 30 to 60, 30 to 40, 40 to 200, 40 to 160, 40 to 140, 40 to 120, 40 to 100, 40 to 80, 40 to 70, 40 to 60, 60 to 200, 60 to 160, 60 to 140, 60 to 120, 60 to 100, 60 to 80, 60 to 70, 70 to 200, 70 to 160, 70 to 140, 70 to 120, 70 to 100, 70 to 80, 80 to 200, 80 to 160, 80 to 140, 80 to 120, 80 to 100, 100 to 200, 100 to 160, 100 to 140, 100 to 120, 120 to 200, 120 to 160, 120 to 140, 140 to 200, 140 to 160, or 160 to 200 nucleotides in length.

In some aspects, the barcode is between about 20 to 200, about 20 to 160, about 20 to 140, about 20 to 120, about 20 to 100, about 20 to 80, about 20 to 70, about 20 to 60, about 20 to 40, about 20 to 30, about 30 to 200, about 30 to 160, about 30 to 140, about 30 to 120, about 30 to 100, about 30 to 80, about 30 to 70, about 30 to 60, about 30 to 40, about 40 to 200, about 40 to 160, about 40 to 140, about 40 to 120, about 40 to 100, about 40 to 80, about 40 to 70, about 40 to 60, about 60 to 200, about 60 to 160, about 60 to 140, about 60 to 120, about 60 to 100, about 60 to 80, about 60 to 70, about 70 to 200, about 70 to 160, about 70 to 140, about 70 to 120, about 70 to 100, about 70 to 80, about 80 to 200, about 80 to 160, about 80 to 140, about 80 to 120, about 80 to 100, about 100 to 200, about 100 to 160, about 100 to 140, about 100 to 120, about 120 to 200, about 120 to 160, about 120 to 140, about 140 to 200, about 140 to 160, or about 160 to 200 nucleotides in length.

In some aspects the one or more polynucleotide sequences of the native barcode polynucleotide cassette are separated by a naturally occurring linker sequence from the native microorganism or from the genome of any microorganism sharing the same genus. In some aspects, the linker sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some aspects, the linker sequence is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides in length. In some aspects the linker sequence is fewer than 2, fewer than 3, fewer than 4, fewer than 5, fewer than 6, fewer than 7, fewer than 8, fewer than 9, fewer than 10, fewer than 11, fewer than 12, fewer than 13, fewer than 14, fewer than 15, fewer than 16, fewer than 17, fewer than 18, fewer than 19, or fewer than 20 nucleotides in length.

In some aspects, the application is drawn to a composition comprising one or more modified cells, wherein at least one of the modifications is the insertion of the native barcode polynucleotide cassette into the cells. In some aspects, the one or more modified cells comprise in their genome (i) a naturally occurring first nucleotide sequence, a naturally occurring second nucleotide sequence, and a naturally occurring third nucleotide sequence; and (ii) a native barcode polynucleotide cassette comprising the first, second, and third nucleotide sequences oriented as 5′ to 3′ as: (1) the first nucleotide sequence, wherein the first nucleotide sequence is a forward primer binding site, (2) the second nucleotide sequence, wherein the second nucleotide sequence is a barcode, and (3) the third nucleotide sequence, wherein the third nucleotide sequence is a reverse primer binding site; wherein the first, second, and third nucleotide sequences of (ii) are in a different proximity to one another as compared to the proximity of the naturally occurring sequences of (i) in their native locations in the genome.

In some aspects, the disclosure is drawn to a method of barcoding a cell. In some aspects, the method of barcoding a cell comprises (a) obtaining a cell; (b) selecting and isolating a first nucleotide sequence in the genome of the cell, selecting and isolating a second nucleotide sequence in the genome of the cell, selecting and isolating a third nucleotide sequence in the genome of the cell; (c) creating a native barcode polynucleotide cassette comprising the first, second, and third nucleotide sequences oriented 5′ to 3′ as: (i) the first nucleotide sequence, wherein the first nucleotide sequence is a forward primer binding site, (ii) the second nucleotide sequence, wherein the second nucleotide sequence is a barcode, and (iii) the third nucleotide sequence, wherein the third nucleotide sequence is a reverse primer binding site; and (d) inserting the native barcode polynucleotide cassette into the genome of a host cell of the same genus or species as the cell in (a); wherein the nucleotide sequences of (i), (ii), and (iii) are native to the host cell.

In some aspects, the disclosure is drawn to a method of screening cells in a sample. In some aspects, the method of screening cells in a sample comprises (a) inserting a native barcode polynucleotide cassette into the genomes of two or more cells, wherein the cassette comprises nucleotide sequences native to each of the two or more cells, oriented 5′ to 3′ as: (i) a forward primer binding site, (ii) a barcode unique to each cell, species or strain, and (iii) a reverse primer binding site; (b) applying the cells to a proliferative medium; (c) collecting a sample of the proliferative medium comprising the cells; and (d) determining the abundance of the cells in the sample by analyzing the abundance of the microbes. The abundance can be a relative abundance or an absolute abundance. The samples can be any sample as provided herein such as, for example, a plant cell. The plant cell can be a non-leguminous plant cell. The plant cell can be from any portion or part of the plant such as, for example, the roots.

In one embodiment, the abundance of cells each comprising a native barcode polynucleotide cassette in their genome is determined in the sample relative to the abundance of each other cell comprising a native barcode polynucleotide cassette in their genome in the sample. Further to this embodiment, the native barcode polynucleotide cassettes present in each of the cells in the sample for which the relative abundance is to be determined comprise the same or identical forward and reverse primer binding sites and different barcode sequences located there between. The relative abundance can be determined via the generation and analysis of sequences reads for each cell comprising a native barcode polynucleotide cassette as provided herein (e.g., RB-TnSeq).

In some cases, the percent relative abundance of a specific native barcode containing strains can be determined by processing raw next generation sequencing (NGS) reads (e.g., NGS reads produced on the Illumina MiSeq platform) for quality and length, identifying barcode sequences within the read, and matching observed barcode sequences to known barcode sequences. The matching of observed barcode sequences to known barcode sequences can be performed using any sequence similarity search program for performing global or local sequence alignment known in the art. In some aspects, the matching is accomplished by employing a program that utilizes the Smith-Waterman algorithm or the Needleman-Wunsch algorithm. Examples of sequence alignment programs for use in the methods provided herein can include BLAST, Bowtie, vsearch, usearch, NW-align, GGSEARCH, GLSEARCH, DNASTAR, JAligner, DNADot, ALLALIGN, ACANA, needle, matcher, NW, water and wordmatch. A comparison between the relative abundance of a specific strain containing a specific native barcode at inoculation vs. the relative abundance on a subsequent harvest date of a plant colonized with the specific strain can be performed in order to highlight colonization capacity of the specific strain. A comparison of the colonization capacity of multiple strains, each labelled with a unique native barcode can then be directly compared in order to determine relative colonization capacities of multiple strains.

In one embodiment, the absolute or total abundance of cells comprising a native barcode polynucleotide cassette in their genome is determined in the sample. The absolute abundance of cells comprising a native barcode polynucleotide cassette in their genome can be determined by generating a control barcode that comprises forward and reverse primer binding sites that are the same or identical to the forward and reverse primer binding sites present in a native barcode polynucleotide cassette in the genome of the cells whose absolute abundance is desired. The control barcode can further comprise a sequence located between said forward and reverse primer binding sites that is unique to the control barcode. In some cases, in order to determine the absolute abundance of cells comprising a native barcode polynucleotide cassette in their genome in the sample, the control DNA corresponding to the native barcode polynucleotide cassette present in the genome of the cells can be spiked into DNA (e.g., genomic DNA) isolated from the sample at a single, two, or a ladder (i.e., 3 or more) of known concentrations or copy numbers in order to generate a standard curve of sequence read counts for a specific native barcode polynucleotide cassette following amplification and sequencing against which the read counts from the cells comprising the corresponding native barcode polynucleotide cassette present in their genome can be compared and their absolute abundance ascertained therefrom. A control barcode or control barcode ladder can be generated for each native barcode polynucleotide present in cells that may be present in a sample. The ladder of known concentrations or copy numbers of any control DNA corresponding to the native barcode polynucleotide cassette present in the genome of cells in a sample can comprise at least 3, 4, 5, 6, 7, 8, 9 or 10 concentrations or copy numbers. The ladder of known concentrations or copy numbers of any control DNA corresponding to the native barcode polynucleotide cassette present in the genome of cells in a sample can comprise at most 3, 4, 3, 6, 7, 8, 9 or 10 concentrations or copy numbers. The two or ladder of control barcodes can be spiked in or added to a DNA preparation at concentrations that span several orders of magnitude. The absolute or total abundance can be expressed as cells per gram of fresh weight (gfw) of a strain. The cell per gfw of a strain can be calculated from amplification and sequencing data generated using any of methods provided herein in combination with using a least-squares linear regression model. In order to determine the cell per gfw of a strain or each of a plurality of strains, the least-squares linear regression model can use read counts of a control barcode (or each of a plurality of control barcodes) and the known copy number of the control barcode (or each of the plurality of control barcodes) to create a function of read counts to number of copies present in the amplification reaction (e.g., PCR reaction) for the strain or strains present in the nucleic acid preparation used for the amplification reaction. The accuracy of the least squares linear regression model in predicting the number of copies of a strain can be determined or measured by calculating the R squared value. The number of copies present in the amplification reaction for the strain or each of the strains from the plurality of strains as determined from the least-squares linear regression model can then be used along with a Tissue Factor (the amount of root tissue sampled relative to the total root tissue present in a plant inoculated with the strain for which the cells per gfw is being calculated), and an elution volume (the amount of liquid used to elute during the gDNA preparations of the sampled root tissue) to calculate the total abundance of the strain or each of the strains from the plurality of strains in the entire root of the plant inoculated with the strain or strains for which the cells per gfw is being calculated. This can be done by multiplying the number of copies calculated to be present in the reaction for a strain by the Tissue Factor and the Elution volume and dividing by the amount of template used as seen in the equation below:

${{cell}\mspace{14mu}{per}\mspace{14mu}{gfw}} = {{copies}\mspace{14mu}{in}\mspace{14mu}{{reaction}\left( \frac{{Tissue}\mspace{14mu}{Factor}*{Elution}\mspace{14mu}{volume}}{{Volume}\mspace{14mu}{of}\mspace{14mu}{template}\mspace{14mu}{used}} \right)}}$

The control barcode or each of the plurality of control barcodes can be sea otter control barcodes as provided herein.

In some aspects, the native barcode polynucleotide cassette is inserted into the genome of the cells by utilizing a vector capable of inserting the cassette. In some aspects, the vector only inserts the native barcode polynucleotide cassette. In some aspects, the vector inserts the native barcode polynucleotide cassette along with one or more flanking sequences from the vector. In some aspects, the one or more flanking sequences from the vector are excised from the genome of the cell, leaving only the native barcode polynucleotide cassette inserted in the genome of the cell. In some aspects, the vector is a suicide vector.

A common issue arising from next-generation sequencing can be bias introduced in sequencing coverage due to distance from or proximity to the origin of replication. Genomic regions near the origin of replication can have a higher copy number in dividing cells and thus can appear more frequently in the sequencing results as described in Wetmore K M, et al., “Rapid Quantification of Mutant Fitness in Diverse Bacteria by Sequencing Randomly Bar-Coded Transposons,” mBio May 2015, 6 (3) e00306-15, which is herein incorporated by reference.

In order to avoid or mitigate this issue, the native barcode polynucleotide sequences provided herein can be designed to be inserted into the genome of a microbial species, strain or variant provided herein such that a semi-constant distance from the origin of replication of the microbial chromosome in each strain is maintained.

In order to determine where to insert a native barcode polynucleotide sequence with respect to an origin of replication, the Ori-Finder (see Gao F, Zhang C-T (2008) Ori-Finder: a web-based system for finding oriCs in unannotated bacterial genomes. BMC Bioinformatics, 9:79 as well as Gao F, Zhang C-T (2007) DoriC: a database of oriC regions in bacterial genomes. Bioinformatics, 23, 1866-1867, each of which are herein incorporated by reference) can be used to first identify the origin of replication sequences in a large public dataset of curated, closed genomes of organisms closely related to a particular strain of interest. These origin of replication sequences can then be assembled into a database and used to identify a likely origin of replication in the strain of interest. Candidate insertion regions for native barcode polynucleotides provided herein can be subsequently considered if they fall within a set distance of an identified origin of replication. The set distance can be any distance from the origin of replication, provided that the barcode is consistently incorporated at the same or similar distance from the origin of replication in each cell in which a barcode is incorporated. Alternative methods to Ori-Finder that can be used for identifying likely origins and termini of replication include calculating the GC skew or Z-curves of (closed) genomes (//en.wikipedia.org/wiki/GC_skew).

Additionally, candidate insertion regions can also be identified by finding a non-coding region between two coding DNA segments which are transcribed in alternate directions and separated by a terminator region (see FIG. 6A). When identifying insertion sites in related strains, homologous regions between strains can be selected via Blast (see Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403-410, which is herein incorporated by reference). As a native barcode polynucleotide sequence complete with universal primer binding sites is placed on one side of the terminator (see FIG. 6B), the transcription of the barcode may only occur in one direction as opposed to two. In addition, stop codons at the end of either gene cease translation of the RNA sequences before a ribosome may encounter the barcode sequence. Furthermore, the greater surrounding genetic context of the region can be screened to ensure no nearby genetic element has proved to be critical in previous studies.

In some aspects, the proliferative medium is a growth medium. In some aspects the proliferative medium is a liquid. In some aspects, the proliferative medium is a solid. In some aspects, the proliferative medium is a semi-solid. In some aspects the proliferative medium is a tissue culture medium. In some aspects the proliferative medium is soil. In some aspects, the soil comprises plants or plant parts. In some aspects, the soil comprises plant roots. In some aspects, the proliferative medium is a medium on/in which the cells grow and divide. In some aspects, the proliferative medium is a medium on/in which the cells are capable of log-phase growth.

In some aspects, there is a period of time between applying the cells to the proliferative medium and collecting a sample of the proliferative medium comprising the cells. In some aspects, the period of time is an amount of time sufficient for the cells to double in number at least once. In some aspects, the period of time is an amount of time sufficient for the cells to double in number at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, or at least 70 times.

In some aspects, the period of time is at least 20 minutes, at least 40 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 17 hours, at least 19 hours, at least 21 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 66 hours, at least 72 hours, at least 3 days, at least 4 days, at least at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days or at least 20 days.

Methods of amplifying and sequencing nucleic acid sequences are well known in the art. In some aspects, the determination of the abundance of the cells requires analyzing the number of barcodes in the samples, which requires amplifying and sequencing the barcode containing samples.

In some embodiments, the amplification is exponential, e.g. in the enzymatic amplification of specific double stranded sequences of DNA by a polymerase chain reaction (PCR). In other embodiments the amplification method is linear. In other embodiments the amplification method is isothermal. In some embodiments, the amplification is exponential, e.g. in the enzymatic amplification of specific double stranded sequences of DNA by a polymerase chain reaction (PCR).

Suitable amplification reactions can be exponential or isothermal and can include any DNA amplification reaction, including but not limited to polymerase chain reaction (PCR), strand displacement amplification (SDA) linear amplification, multiple displacement amplification (MDA), rolling circle amplification (RCA), single primer isothermal amplification (SPIA, see e.g. U.S. Pat. No. 6,251,639), Ribo-SPIA, or a combination thereof. In some cases, the amplification methods for providing the template nucleic acid may be performed under limiting conditions such that only a few rounds of amplification (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, 30 etc.), such as, for example, as is commonly done for cDNA generation. The number of rounds of amplification can be about 1-30, 1-20, 1-10, 5-30, 10-30, 15-30, 20-30, 10-30, 15-30, 20-30, or 25-30.

PCR is an in vitro amplification procedure based on repeated cycles of denaturation, oligonucleotide primer annealing, and primer extension by thermophilic template dependent polynucleotide polymerase, resulting in the exponential increase in copies of the desired sequence of the polynucleotide analyte flanked by the primers. The two different PCR primers, which anneal to opposite strands of the DNA, are positioned so that the polymerase catalyzed extension product of one primer can serve as a template strand for the other, leading to the accumulation of a discrete double stranded fragment whose length is defined by the distance between the 5′ ends of the oligonucleotide primers. PCR reactions suitable for the methods of the present disclosure include real-time PCR, multiplex PCR, quantitative PCR, digital PCR, and digital droplet PCR.

Randomly Barcoded Transposon Sequencing (RB-TnSeq)

Randomly barcoded transposon sequencing (RB-TnSeq) is an important tool to characterize genes in cultivable microorganisms using phenotype data (Wetmore et al (2015) Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly barcoded transposons (RB-TnSeq). mBio 6(3):e00306-15).

High-throughput mutant fitness profiling in bacteria is commonly performed by mixing a large number of transposon mutants and monitoring their abundance in a competitive growth assay with next-generation sequencing. A number of approaches have been developed for transposon insertion site sequencing in bacteria and they may be collectively referred to as TnSeq. Typically, in TnSeq, genomic DNA (gDNA) at the transposon insertion site serves as the “tag” for identifying each strain, and the importance of each gene for fitness is estimated from the number of reads that correspond to insertions within that gene.

For instance, one Tn-seq method uses the Himar I mariner transposon, a common and stable transposon. Through a single nucleotide change in the terminal repeats of mariner, the recognition sites for a type IIS restriction endonuclease called MmeI can be introduced into the transposon. MmeI makes a 2 base pair staggered cut 20 bases downstream of the recognition site. DNA extracted from a library of transposon insertion mutants is digested with MmeI. Upon digestion, fragmented DNA including the 16 base pairs of the surrounding genomic DNA is produced. The 16 base pair fragment can be used to determine the location of the transposon insertion in the bacterial genome by sequencing. However, the mariner transposon inserts only at TA sites, therefore this method is not ideal for genomes with high GC content. Therefore the TnSeq protocol has been updated to be used with any transposon; yet, DNA shearing and multiple enzymatic steps are still required. Thus, even though mutant fitness can be determined via TnSeq, this technique and variations thereof have not been applied to hundreds of experimental conditions likely because, the preparation of TnSeq sequencing libraries is laborious.

In RB-TnSeq, DNA barcoded transposons are used to generate large bacterial mutant populations with the aim that each mutant strain in the population carries a single transposon insertion containing a unique DNA barcode. To identify phenotypes and gain insight into gene function, bar-coded transposon mutant populations are subject to competitive growth assays. In these experiments, the relative abundance of each mutant strain changes depending on the impact of the underlying gene mutation on the fitness of that strain. The developers of RB-TnSeq converted Tn5 and mariner transposon delivery vectors into RB-TnSeq vectors by cloning millions of random 20-nucleotide DNA barcodes near the edge of each transposon's inverted repeat. Using these transposons, genome-wide transposon mutant libraries were generated in five bacteria. For each mutant library, TnSeq was performed to characterize the mutant library by simultaneously mapping the transposon insertion location and the identity of the linked DNA barcode in a single Illumina read. Over 100,000 transposon insertions with unique DNA barcodes were identified in each of the five mutant libraries. Subsequently, BarSeq, or deep sequencing of DNA barcodes, was used to quantify the abundance of transposon insertion strains and calculate gene fitness in a competitive growth assay. That is, to determine the fitness of each gene under a given condition, the abundance of the barcodes for different strains of a gene (independent transposon insertions) were compared before and after growth selection.

However, even with RB-TnSeq, it can be challenging to infer gene functions without a significant loss-of-function phenotype due to single mutations. Cultivating and mutagenizing microbial isolates from the field is not feasible with RB-TnSeq if the goal is to eventually utilize these mutants in the field, because of the limits on applying transgenic strains in the environment.

16S-Based Metagenomic Profiling

As used herein, the term “metagenome” refers to genomic material obtained directly from a microbe or microbial population. As used herein, the term “metagenomics” refers to the application of modern genomics techniques to the study of communities of microbial organisms directly in their environment based on DNA obtained from a sample. Metagenomics provides information regarding the genomic material of a microbial community.

Metagenomics allows the direct genetic analysis of genomes contained within an environmental sample without the prior need for cultivating clonal cultures through the use of next-generation sequencing (NGS) or high-throughput sequencing. A metagenomics analysis might involve functional and sequence-based analysis of the collective microbial genomes contained in an environmental sample, referred to as shotgun metagenomics. The shotgun sequencing approach, which was used to sequence many cultured microorganisms and the human genome, has been adapted to the analysis of DNA sequences recovered from environmental samples. Shotgun metagenomics has the capacity to fully sequence the majority of available genomes within an environmental sample (or community).

Alternatively, a metagenomics analysis might involve polymerase chain reaction (PCR) amplification of certain “marker” genes of interest, such as 16S ribosomal RNA gene, and sequencing of these marker genes. This metagenomics approach may be referred to as gene-based metagenomics or in the case of 16S rRNA, 16S rRNA-based metagenomics. The 16S ribosomal RNA (or 16S rRNA) is one of the non-coding RNA molecules present within the microbial ribosome. 16S rRNA gene fragments can also be retrieved from shotgun metagenomic sequences (metagenomes) and used for species profiling. Both approaches have their limitations. 16S rRNA sequencing may be biased because of unequal amplification of species' 16S rRNA genes, whereas shotgun metagenomic sequencing may not be deep enough to detect the 16S rRNA genes of rare species in a community.

Broad-range PCR primers targeted to highly conserved regions of the 16S rRNA gene enables the amplification of rRNA gene sequences from all bacterial species; and the extensive and rapidly growing 16S gene sequence database facilitates identification of sequences to the species or genus level. Bacterial 16S ribosomal RNA (rRNA) genes contain nine “hypervariable regions” (VI-V9) that demonstrate considerable sequence diversity among different bacteria. A disadvantage associated with relying on a hypervariable region to identify bacteria in a sample is that 16S rRNA hypervariable regions exhibit different degrees of sequence diversity, and no single hypervariable region is able to distinguish among all bacteria. Theoretically, the full-length 16S rRNA genes (about 1500 bp) can be used for accurate taxonomic identification based on the underlying sequence diversity among different bacterial species. However, the current high-throughput DNA sequencing technologies can only produce 16S rRNA gene fragments, instead of full-length genes. Therefore, obtaining reliable representation of bacterial communities through taxonomic classification of short 16S rRNA gene sequences can be challenging.

Transcriptomic Profiling of Candidate Microbes

Previous work by the inventors entailed transcriptomic profiling of strain CIO 10 to identify promoters that are active in the presence of environmental nitrogen. Strain CI010 was cultured in a defined, nitrogen-free media supplemented with 10 mM glutamine. Total RNA was extracted from these cultures (QIAGEN RNeasy kit) and subjected to RNAseq sequencing via Illumina HiSeq (SeqMatic, Fremont Calif.). Sequencing reads were mapped to the CIO 10 genome data using Geneious, and highly expressed genes under control of proximal transcriptional promoters were identified.

Tables 21-23 lists genes and their relative expression level as measured through RNASeq sequencing of total RNA. Sequences of the proximal promoters were recorded for use in mutagenesis of nif pathways, nitrogen utilization related pathways, or other genes with a desired expression level.

TABLE 21 Name Minimum Maximum Length Direction murein lipoprotein CDS 2,929,898 2,930,134 237 forward membrane protein CDS 5,217,517 5,217,843 327 forward zinc/cadmium-binding 3,479,979 3,480,626 648 forward protein CDS acyl carrier protein CDS 4,563,344 4,563,580 237 reverse ompX CDS 4,251,002 4,251,514 513 forward DNA-binding protein 375,156 375,428 273 forward HU-beta CDS sspA CDS 629,998 630,636 639 reverse taffi CDS 3,199,435 3,199,638 204 reverse LexA repressor CDS 1,850,457 1,851,065 609 forward hisS CDS <3999979 4,001,223 >1245 forward

TABLE 22 Differential RNASeq_(—) RNASeq_(—) RNASeq_(—) RNASeq_(—) Expression Differential nifL - Raw nifL - Raw WT - Raw WT - Raw Absolute Expression Read Transcript Read Transcript Name Confidence Ratio Count Count Count Count murein 1000 −1.8 12950.5 10078.9 5151.5 4106.8 lipoprotein CDS membrane 1000 −1.3 9522.5 5371.3 5400 3120 protein CDS zinc/cadmium- 3.3 1.1 6461 1839.1 5318 1550.6 binding protein CDS acyl carrier 25.6 1.6 1230.5 957.6 1473.5 1174.7 protein CDS ompX CDS 1.7 1.1 2042 734.2 1687.5 621.5 DNA-binding 6.9 −1.3 1305 881.7 725 501.8 protein HU- beta CDS sspA CDS 0.2 1 654 188.8 504.5 149.2 tatE CDS 1.4 1.3 131 118.4 125 115.8 LexA 0.1 −1.1 248 75.1 164 50.9 repressor CDS hisS CDS 0 −1.1 467 69.2 325 49.3

TABLE 23 Prm (In Forward direction, −250 Expressed Neighbor to +10 region) Sequence Sequence Name SEQ ID NO: SEQ ID NO: SEQ ID NO: murein lipoprotein CDS SEQ ID NO: 3 SEQ ID NO: 13 SEQ ID NO: 23 membrane protein CDS SEQ ID NO: 4 SEQ ID NO: 14 SEQ ID NO: 24 zinc/cadmium-binding SEQ ID NO: 5 SEQ ID NO: 15 SEQ ID NO: 25 protein CDS acyl carrier protein CDS SEQ ID NO: 6 SEQ ID NO: 16 SEQ ID NO: 26 ompX CDS SEQ ID NO: 7 SEQ ID NO: 17 SEQ ID NO. 27 DNA-binding protein SEQ ID NO: 8 SEQ ID NO: 18 SEQ ID NO: 28 HU-beta CDS sspA CDS SEQ ID NO: 9 SEQ ID NO: 19 SEQ ID NO: 29 tatE CDS SEQ ID NO: 10 SEQ ID NO: 20 SEQ ID NO: 30 LexA repressor CDS SEQ ID NO: 11 SEQ ID NO: 21 SEQ ID NO: 31 hisS CDS SEQ ID NO: 12 SEQ ID NO: 22 SEQ ID NO: 32

TABLE 24 Table of Strains Mutagenic DNA Gene 1 Gene 2 Name Lineage Description Genotype mutation mutation CI006 Isolated strain from None WT Enterobacter (now Kosakonia) genus CI008 Isolated strain from None WT Burkholderia (now Paraburkholderia) genus CI010 Isolated strain from None WT Klebsiella genus CI019 Isolated strain from None WT Rahnella genus CI028 Isolated strain from None WT Enterobacter genus CI050 Isolated strain from None WT Klebsiella genus CM002 Mutant of C1050 Disruption of nifL gene ΔnifL::KanR SEQ ID with a kanamycin resistance NO: 33 expression cassette (KanR) encoding the aminoglycoside O- phosphotransferase gene aph1 inserted. CM011 Mutant of CI019 Disruption of nifL gene ΔnifL::SpecR SEQ ID with a spectinomycin NO: 34 resistance expression cassette (SpecR) encoding the streptomycin 3″-O- adenylyltransferase gene aadA inserted. CM013 Mutant of CI006 Disruption of nifL gene ΔnifL::KanR SEQ ID with a kanamycin resistance NO: 35 expression cassette (KanR) encoding the aminoglycoside O- phosphotransferase gene aph1 inserted. CM004 Mutant of CI010 Disruption of amtB gene ΔamtB::KanR SEQ ID with a kanamycin resistance NO: 36 expression cassette (KanR) encoding the aminoglycoside O- phosphotransferase gene aph1 inserted. CM005 Mutant of CI010 Disruption of nifL gene ΔnifL::KanR SEQ ID with a kanamycin resistance NO: 37 expression cassette (KanR) encoding the aminoglycoside O- phosphotransferase gene aph1 inserted. CM015 Mutant of CI006 Disruption of nifL gene ΔnifL::Prm5 SEQ ID with a fragment of the NO: 38 region upstream of the ompX gene inserted (Prm5). CM021 Mutant of CI006 Disruption of nifL gene ΔnifL::Prm2 SEQ ID with a fragment of the NO: 39 region upstream of an unanotated gene and the first 73 bp of that gene inserted (Prm2). CM023 Mutant of CI006 Disruption of nifL gene ΔnifL::Prm4 SEQ ID with a fragm ent of the NO: 40 region upstream of the acpP gene and the first 121 bp of the acpP gene inserted (Prm4). CM014 Mutant of CI006 Disruption of nifL gene ΔnifL::Prm1 SEQ ID with a fragment of the NO: 41 region upstream of the lpp gene and the first 29 bp of the lpp gene inserted (Prm1). CM016 Mutant of CI006 Disruption of nifL gene ΔnifL::Prm9 SEQ ID with a fragment of the NO: 42 region upstream of the lexA 3 gene and the first 21 bp of the lexA 3 gene inserted (Prm9). CM022 Mutant of C1006 Disruption of nifL gene ΔnifL::Prm3 SEQ ID with a fragment of the NO: 43 region upstream of the mntP 1 gene and the first 53 bp of the mntP 1 gene inserted (Prm3). CM024 Mutant of CI006 Disruption of nifL gene ΔnifL::Prm7 SEQ ID with a fragment of the NO: 44 region upstream of the sspA gene inserted (Prm7). CM025 Mutant of CI006 Disruption of nifL gene ΔnifL::Prm10 SEQ ID with a fragment of the NO: 45 region upstream of the hisS gene and the first 52 bp of the hisS gene inserted (Prm10). CM006 Mutant of CI010 Disruption of glnB gene ΔglnB::KanR SEQ ID with a kanamycin resistance NO: 46 expression cassette (KanR) encoding the aminoglycoside O- phosphotransferase gene aph1 inserted. CM017 Mutant of CI028 Disruption of nifL gene ΔnifL::KanR SEQ ID with a kanamycin resistance NO: 47 expression cassette (KanR) encoding the aminoglycoside O- phosphotransferase gene aph1 inserted. CM011 Mutant of CI019 Disruption of nifL gene ΔnifL::SpecR SEQ ID with a spectinomycin NO: 48 resistance expression cassette (SpecR) encoding the streptomycin 3″-O- adenylyltransferase gene aadA inserted. CM013 Mutant of CI006 Disruption of nifL gene ΔnifL:: KanR SEQ ID with a kanamycin resistance NO: 49 expression cassette (KanR) encoding the aminoglycoside O- phosphotransferase gene aph1 inserted. CM005 Mutant of CI010 Disruption of nifL gene ΔnifL::KanR SEQ ID with a kanamycin resistance NO: 50 expression cassette (KanR) encoding the aminoglycoside O- phosphotransferase gene aph1 inserted. CM014 Mutant of CI006 Disruption of nifL gene ΔnifL::Prml SEQ ID with a fragment of the NO: 51 region upstream of the lpp gene and the first 29 bp of the lpp gene inserted (Prm1). CM015 Mutant of CI006 Disruption of nifL gene ΔnifL::Prm5 SEQ ID with a fragment of the NO: 52 region upstream of the ompX gene inserted (Pnn5). CM023 Mutant of CI006 Disruption of nifL gene ΔnifL::Prm4 SEQ ID with a fragment of the NO: 53 region upstream of the acpP gene and the first 121 bp of the acpP gene inserted (Prm4). CM029 Mutant of CI006 Disruption of nifL gene ΔnifL::Prm5 SEQ ID SEQ ID with a fragment of the ΔglnE- NO: 54 NO: 61 region upstream of the AR_KO1 ompX gene inserted (Prm5) and deletion of the 1287 bp after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate-ammonia- ligase adenylyltransferase (ΔglnE-AR_KO1). CM014 Mutant of CI006 Disruption of nifL gene ΔnifL::Prm1 SEQ ID with a fragment of the NO: 55 region upstream of the lpp gene and the first 29 bp of the lpp gene inserted (Prm1). CM011 Mutant of CI019 Disruption of nifL gene ΔnifL::SpecR SEQ ID with a spectinomycin NO: 56 resistance expression cassette (SpecR) encoding the streptomycin 3″-O- adenylyltransferase gene aadA inserted. CM011 Mutant of CI019 Disruption of nifL gene ΔnifL::SpecR SEQ ID with a spectinomycin NO: 57 resistance expression cassette (SpecR) encoding the streptomycin 3″-O- adenylyltransferase gene aadA inserted. CM013 Mutant of CI006 Disruption of nifL gene ΔnifL::KanR SEQ ID with a kanamycin resistance NO: 58 expression cassette (KanR) encoding the aminoglycoside O- phosphotransferase gene aph1 inserted. CM011 Mutant of CI019 Disruption of nifL gene ΔnifL::SpecR SEQ ID with a spectinomycin NO: 59 resistance expression cassette (SpecR) encoding the streptomycin 3″-O- adenylyltransferase gene aadA inserted. CM011 Mutant of CI019 Disruption of nifL gene ΔnifL::SpecR SEQ ID with a spectinomycin NO: 60 resistance expression cassette (SpecR) encoding the streptomycin 3″-O- adenylyltransferase gene aadA inserted.

EXAMPLES

The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. Changes therein and other uses which are encompassed within the spirit of the disclosure, as defined by the scope of the claims, will be recognized by those skilled in the art.

Example 1: Guided Microbial Remodeling—A Platform for the Rational Improvement of Microbial Species for Agriculture

An example overview of an embodiment of the Guided Microbial Remodeling (GMR) platform can be summarized in the schematic of FIG. 1A.

FIG. 1A illustrates that the composition of the microbiome can first be characterized and a species of interest is identified (e.g. to find a microbe with the appropriate colonization characteristics).

The metabolism of the species of interest can be mapped and linked to genetics. For example, the nitrogen fixation pathway of the microbe can be characterized. The pathway that is being characterized can be examined under a range of environmental conditions. For example, the microbe's ability to fix atmospheric nitrogen in the presence of various levels of exogenous nitrogen in its environment can be examined. The metabolism of nitrogen can involve the entrance of ammonia (NH₄ ⁺) from the rhizosphere into the cytosol of the bacteria via the AmtB transporter. Ammonia and L-glutamate (L-Glu) are catalyzed by glutamine synthetase and ATP into glutamine. Glutamine can lead to the formation of bacterial biomass and it can also inhibit expression of the nif operon, i.e. it can be a competing force when one desires the microbe to fix atmospheric nitrogen and excrete ammonia. The nitrogen fixation pathway is characterized in great detail in earlier sections of the specification.

Afterwards, a targeted non-intergeneric genomic alteration can be introduced to the microbe's genome, using methods including, but not limited to: conjugation and recombination, chemical mutagenesis, adaptive evolution, and gene editing. The targeted non-intergeneric genomic alteration can include an insertion, disruption, deletion, alteration, perturbation, modification, etc. of the genome.

Derivative remodeled microbes, which comprise the desired phenotype resulting from the remodeled underlying genotype, are then used to inoculate crops.

The present disclosure provides, in certain embodiments, non-intergeneric remodeled microbes that are able to fix atmospheric nitrogen and supply such nitrogen to a plant. In aspects, these non-intergeneric remodeled microbes are able to fix atmospheric nitrogen, even in the presence of exogenous nitrogen.

FIG. 1B depicts an expanded view of the measurement of the microbiome step. In some embodiments, the present disclosure finds microbial species that have desired colonization characteristics, and then utilizes those species in the subsequent remodeling process.

The aforementioned Guided Microbial Remodeling (GMR) platform will now be described with more specificity.

In aspects, the GMR platform comprises the following steps:

-   -   A. Isolation—Obtain microbes from the soil, rhizosphere,         surface, etc. of a crop plant of interest;     -   B. Characterization—Involves characterizing the isolated         microbes for genotype/phenotypes of interest (e.g. genome         sequence, colonization ability, nitrogen fixation activity,         solubilization of P ability, excretion of a metabolite of         interest, excretion of a plant promoting compound, etc.);     -   C. Domestication—Development of a molecular protocol for         non-intergeneric genetic modification of the microbe;     -   D. Non-intergeneric Engineering Campaign and         Optimization—Generation of derivative non-intergeneric microbial         strains with genetic modifications in key pathways (e.g.         colonization associated genes, nitrogen fixation/assimilation         genes, P solubilization genes);     -   E. Analytics—Evaluation of derived non-intergeneric strains for         phenotypes of interest both in vitro (e.g. ARA assays) and in         planta (e.g. colonization assays);     -   F. Iterate Engineering Campaign/Analytics—Iteration of steps D         and E for further improvement of microbial strain.

Each of the GMR platform process steps will now be elaborated upon below.

A. Isolation of Microbes

1. Obtain a Soil Sample

Microbes will be isolated from soil and/or roots of a plant. In one example, plants will be grown in a laboratory or a greenhouse in small pots. Soil samples will be obtained from various agricultural areas. For example, soils with diverse texture characteristics can be collected, including loam (e.g. peaty clay loam, sandy loam), clay soil (e.g. heavy clay, silty clay), sandy soil, silty soil, peaty soil, chalky soil, and the like.

2. Grow Bait Plants

Seeds of a bait plant (a plant of interest) (e.g. corn, wheat, rice, sorghum, millet, soybean, vegetables, fruits, etc.) will be planted into each soil type. In one example, different varieties of a bait plant will be planted in various soil types. For example, if the plant of interest is corn, seeds of different varieties of corn such as field corn, sweet corn, heritage corn, etc. will be planted in various soil types described above.

3. Harvest Soil and/or Root Samples and Plate on Appropriate Medium

Plants will be harvested by uprooting them after a few weeks (e.g. 2-4 weeks) of growth. Alternative to growing plants in a laboratory/greenhouse, soil and/or roots of the plant of interest can be collected directly from the fields with different soil types.

To isolate rhizosphere microbes and epiphytes, plants will be removed gently by saturating the soil with distilled water or gently loosening the soil by hand to avoid damage to the roots. If larger soil particles are present, these particles will be removed by submerging the roots in a still pool of distilled water and/or by gently shaking the roots. The root will be cut and a slurry of the soil sticking to the root will be prepared by placing the root in a plate or tube with small amount of distilled water and gently shaking the plate/tube on a shaker or centrifuging the tube at low speed. This slurry will be processed as described below.

To isolate endophytes, excess soil on root surfaces will be removed with deionized water. Following soil removal, plants will be surface sterilized and rinsed vigorously in sterile water. A cleaned, 1 cm section of root will be excised from the plant and placed in a phosphate buffered saline solution containing 3 mm steel beads. A slurry will be generated by vigorous shaking of the solution with a Qiagen TissueLyser II.

The soil and/or root slurry can be processed in various ways depending on the desired plant-beneficial trait of microbes to be isolated. For example, the soil and root slurry can be diluted and inoculated onto various types of screening media to isolate rhizospheric, endophytic, epiphytic, and other plant-associated microbes. For example, if the desired plant-beneficial trait is nitrogen fixation, then the soil/root slurry will be plated on a nitrogen free media (e.g. Nfb agar media) to isolate nitrogen fixing microbes. Similarly, to isolate phosphate solubilizing bacteria (PSB), media containing calcium phosphate as the sole source of phosphorus can be used. PSB can solubilize calcium phosphate and assimilate and release phosphorus in higher amounts. This reaction is manifested as a halo or a clear zone on the plate and can be used as an initial step for isolating PSB.

4. Pick Colonies, Purify Cultures, and Screen for the Presence of Genes of Interest

Populations of microbes obtained in step A3 are streaked to obtain single colonies (pure cultures). A part of the pure culture is resuspended in a suitable medium (e.g. a mixture of R2A and glycerol) and subjected to PCR analysis to screen for the presence of one or more genes of interest. For example, to identify nitrogen fixing bacteria (diazotrophs), purified cultures of isolated microbes can be subjected to a PCR analysis to detect the presence of nif genes that encode enzymes involved in the fixation of atmospheric nitrogen into a form of nitrogen available to living organisms.

5. Bank a Purified Culture

Purified cultures of isolated strains will be stored, for example at −80° C., for future reference and analysis.

B. Characterization of Isolated Microbes

1. Phylogenetic Characterization and Whole Genome Sequencing

Isolated microbes will be analyzed for phylogenetic characterization (assignment of genus and species) and the whole genome of the microbes will be sequenced.

For phylogenetic characterization, 16S rDNA of the isolated microbe will be sequenced using degenerate 16S rDNA primers to generate phylogenetic identity. The 16S rDNA sequence reads will be mapped to a database to initially assign the genus, species and strain name for isolated microbes. Whole genome sequencing is used as the final step to assign phylogenetic genus/species to the microbes.

The whole genome of the isolated microbes will be sequenced to identify key pathways. For the whole genome sequencing, the genomic DNA will be isolated using a genomic DNA isolation kit (e.g. QIAmp DNA mini kit from QIAGEN) and a total DNA library will be prepared using the methods known in the art. The whole genome will be sequenced using high throughput sequencing (also called Next Generation Sequencing) methods known in the art. For example, Illumina, Inc., Roche, and Pacific Biosciences provide whole genome sequencing tools that can be used to prepare total DNA libraries and perform whole genome sequencing.

The whole genome sequence for each isolated strain will be assembled; genes of interest will be identified; annotated, and noted as potential targets for remodeling. The whole genome sequences will be stored in a database.

2. Assay the Microbe for Colonization of a Host Plant in a Greenhouse

Isolated microbes will be characterized for the colonization of host plants in a greenhouse. For this, seeds of the desired host plant (e.g., corn, wheat, rice, sorghum, soybean) will be inoculated with cultures of isolated microbes individually or in combination and planted into soil. Alternatively, cultures of isolated microbes, individually or in combination, can be applied to the roots of the host plant by inoculating the soil directly over the roots. The colonization potential of the microbes will be assayed, for example, using a quantitative PCR (qPCR) method described in a greater detail below.

3. Assay the Microbe for Colonization of the Host Plant in Small-Scale Field Trials and Isolate RNA from Colonized Root Samples (CAT Trials)

Isolated microbes will be assessed for colonization of the desired host plant in small-scale field trials. Additionally, RNA will be isolated from colonized root samples to obtain transcriptome data for the strain in a field environment. These small-scale field trials are referred to herein as CAT (Colonization and Transcript) trials, as these trials provide Colonization and Transcript data for the strain in a field environment.

For these trials, seeds of the host plant (e.g., corn, wheat, rice, sorghum, soybean) will be inoculated using cultures of isolated microbes individually or in combination and planted into soil. Alternatively, cultures of isolated microbes, individually or in combination, can be applied to the roots of the host plant by inoculating the soil directly over the roots. The CAT trials can be conducted in a variety of soils and/or under various temperature and/or moisture conditions to assess the colonization potential and obtain transcriptome profile of the microbe in various soil types and environmental conditions.

Colonization of roots of the host plant by the inoculated microbe(s) will be assessed, for example, using a qPCR method as described below.

In one protocol, the colonization potential of isolated microbes was assessed as follows. One day after planting of corn seeds, 1 ml of microbial overnight culture (SOB media) was drenched right at the spot of where the seed was located. 1 mL of this overnight culture was roughly equivalent to about 10{circumflex over ( )}9 cfu, varying within 3-fold of each other, depending on which strain is being used. Each seedling was fertilized 3× weekly with 50 mL modified Hoagland's solution supplemented with either 2.5 mM or 0.25 mM ammonium nitrate. At four weeks after planting, root samples were collected for DNA extraction. Soil debris were washed away using pressurized water spray. These tissue samples were then homogenized using QIAGEN Tissuelyzer and the DNA was then extracted using QIAmp DNA Mini Kit (QIAGEN) according to the recommended protocol. qPCR assay was performed using Stratagene Mx3005P RT-PCR on these DNA extracts using primers that were designed (using NCBI's Primer BLAST) to be specific to a loci in each of the microbe's genome.

The presence of the genome copies of the microbe was quantified, which reflected the colonization potential of the microbe. Identity of the microbial species was confirmed by sequencing the PCR amplification products.

Additionally, RNA will be isolated from colonized root and/or soil samples and sequenced.

Unlike the DNA profile, an RNA profile varies depending on the environmental conditions. Therefore, sequencing of RNA isolated from colonized roots and/or soil will reflect the transcriptional activity of genes in planta in the rhizosphere.

RNA can be isolated from colonized root and/or soil samples at different time points to analyze the changes in the RNA profile of the colonized microbe at these time points.

For example, RNA can be isolated from colonized root and/or soil samples right after fertilization of the field and a few weeks after fertilization of the field and sequenced to generate corresponding transcriptional profile.

Similarly, RNA sequencing can be carried out under high phosphate and low phosphate conditions to understand which genes are transcriptionally active or repressed under these conditions.

Methods for transcriptomic/RNA sequencing are known in the art. Briefly, total RNA will be isolated from the purified culture of the isolated microbe; cDNA will be prepared using reverse transcriptase; and the cDNA will be sequenced using high throughput sequencing tools described above.

Sequencing reads from the transcriptome analysis can be mapped to the genomic sequence and transcriptional promoters for the genes of interest can be identified.

4. Assay the Plant-Beneficial Activity of Isolated Microbes

The plant-beneficial activity of isolated microbes will be assessed.

For example, nitrogen fixing microbes will be assayed for nitrogen fixation activity using an acetylene reduction assay (ARA) or phosphate solubilizing microbes will be assayed for phosphate solubilization. Any parameter of interest can be utilized and an appropriate assay developed for such. For instance, assays could include growth curves for colonization metrics and assays for production of phytohormones like indole acetic acid (IAA) or gibberellins. An assay for any plant-beneficial activity that is of interest can be developed.

This step will confirm the phenotype of interest and eliminate any false positives.

5. Selection of Potential Candidates from Isolated Microbes

The data generated in the above steps will be used to select microbes for further development. For example, microbes showing a desired combination of colonization potential, plant-beneficial activity, and/or relevant DNA and RNA profile will be selected for domestication and remodeling.

C. Domestication of Selected Microbes

The selected microbes will be domesticated; wherein, the microbes will be converted to a form that is genetically tractable and identifiable.

1. Test for Antibiotic Sensitivity

One way to domesticate the microbes is to engineer them with antibiotic resistance. For this, the wild type microbial strain will be tested for sensitivity to various antibiotics. If the strain is sensitive to the antibiotic, then the antibiotic can be a good candidate for use in genetic tools/vectors for remodeling the strain.

2. Design and Build a Vector

Vectors that are conditional for their replication (e.g. a suicide plasmid) will be constructed to domesticate the selected microbes (host microbes). For example, a suicide plasmid containing an appropriate antibiotic resistance marker, a counter selectable marker, an origin of replication for maintenance in a donor microbe (e.g. E. coli), a gene encoding a fluorescent protein (GFP, RFP, YFP, CFP, and the like) to screen for insertion through fluorescence, an origin of transfer for conjugation into the host microbe, and a polynucleotide sequence comprising homology arms to the host genome with a desired genetic variation will be constructed. The vector may comprise a SceI site and other additional elements.

Exemplary antibiotic resistance markers include ampicillin resistance marker, kanamycin resistance marker, tetracycline resistance marker, chloramphenicol resistance marker, erythromycin resistance marker, streptomycin resistance marker, spectinomycin resistance marker, etc. Exemplary counter selectable markers include sacB, rpsL, tetAR, pheS, thyA, lacY, gata-1, ccdB, etc.

3. Generation of Donor Microbes

In one protocol, a suicide plasmid containing an appropriate antibiotic resistance marker, a counter selectable marker, the λpir origin of replication for maintenance in E. coli ST18 containing the pir replication initiator gene, a gene encoding green fluorescent protein (GFP) to screen for insertion through fluorescence, an origin of transfer for conjugation into the host microbe, and a polynucleotide sequence comprising homology arms to the host genome with a desired genetic variation (e.g. a promoter from within the microbe's own genome for insertion into a heterologous location) will be transformed into E. coli ST18 (an auxotroph for aminolevulinic acid, ALA) to generate donor microbes.

4. Mix Donor Microbes with Host Microbes

Donor microbes will be mixed with host microbes (selected candidate microbes from step B5) to allow conjugative integration of the plasmid into the host genome. The mixture of donor and host microbes will be plated on a medium containing the antibiotic and not containing ALA. The suicide plasmid is able to replicate in donor microbes (E. coli ST18), but not in the host. Therefore, when the mixture containing donor and host microbes is plated on a medium containing the antibiotic and not containing ALA, only host cells that integrated the plasmid into its genome will be able to grow and form colonies on the medium. The donor microbes will not grow due to the absence of ALA.

5. Confirm Integration of the Vector

A proper integration of the suicide plasmid containing the fluorescent protein marker, the antibiotic resistance marker, the counter selectable marker, etc. at the intended locus of the host microbe will be confirmed through fluorescence of colonies on the plate and using colony PCR.

6. Streak Confirm Integration Colony

A second round of homologous recombination in the host microbes will loop out (remove) the plasmid backbone leaving the desired genetic variation (e.g. a promoter from within the microbe's own genome for insertion into a heterologous location) integrated into the host genome of a certain percentage of host microbes, while reverting a certain percentage back to wild type.

Colonies of host microbes that have looped out the plasmid backbone (and therefore, looped out the counter selectable marker) can be selected by growing them on an appropriate medium.

For example, if sacB is used as a counter selectable marker, loss of this marker due to the loss of the plasmid backbone will be tested by growing the colonies on a medium containing sucrose (sacB confers sensitivity to sucrose). Colonies that grow on this medium would have lost the sacB marker and the plasmid backbone and would either contain the desired genetic variation or be reverted to wild type. Also, these colonies will not fluoresce on the plate due to the loss of the fluorescent protein marker.

In some isolates, the sacB or other counterselectable markers do not confer full sensitivity to sucrose or other counterselection mechanisms, which necessitates screening large numbers of colonies to isolate a successful loop-out. In those cases, loop-out may be aided by use of a “helper plasmid” that replicates independently in the host cell and expresses a restriction endonuclease, e.g. SceI, which recognizes a site in the integrated suicide plasmid backbone. The strain with the integrated suicide plasmid is transformed with the helper plasmid containing an antibiotic resistance marker, an origin of replication compatible with the host strain, and a gene encoding a restriction endonuclease controlled by a constitutive or inducible promoter. The double-strand break induced in the integrated plasmid backbone by the restriction endonuclease promotes homologous recombination to loop-out the suicide plasmid. This increases the number of looped-out colonies on the counterselection plate and decreases the number of colonies that need to be screened to find a colony containing the desired mutation. The helper plasmid is then removed from the strain by culture and serial passaging in the absence of antibiotic selection for the plasmid. The passaged cultures are streaked for single colonies, colonies are picked and screened for sensitivity to the antibiotic used for selection of the helper plasmid, as well as absence of the plasmid confirmed by colony PCR. Finally, the genome is sequenced and the absence of helper plasmid DNA is confirmed as described in D6.

7. Confirm Integration of the Genetic Variation Through Colony PCR

The colonies that grew better on the sucrose-containing medium (or other appropriate media depending on the counter selectable marked used) will be picked and the presence of the genetic variation at the intended locus will be confirmed by screening the colonies using colony PCR

Although this example describes one protocol for domesticating the microbe and introducing genetic variation into the microbe, one of ordinary skill in the art would understand that the genetic variation can be introduced into the selected microbes using a variety of other techniques known in the art such as: polymerase chain reaction mutagenesis, oligonucleotide-directed mutagenesis, saturation mutagenesis, fragment shuffling mutagenesis, homologous recombination, ZFN, TALENS, CRISPR systems (Cas9, Cpf1, etc.), chemical mutagenesis, and combinations thereof.

8. Iterate Upon Steps C2-C7

If any of the steps C2-C7 fail to provide the intended outcome, the steps will be repeated to design an alternative vector that may comprise different elements for facilitating incorporation of desired genetic variations and markers into the host microbe.

9. Develop a Standard Operating Procedure (SOP)

Once the steps C2-C7 can be reproduced consistently for a given strain, the steps will be used to develop a standard operating procedure (SOP) for that strain and vector. This SOP can be used to improve other plant-beneficial traits of the microbe.

D. Non-Intergeneric Engineering Campaign and Optimization

1. Identify Gene Targets for Optimization

Selected microbes will be engineered/remodeled to improve performance of the plant-beneficial activity. For this, gene targets for improving the plant-beneficial activity will be identified.

Gene targets can be identified in various ways. For example, genes of interest can be identified while annotating the genes from the whole genome sequencing of isolated microbes. They can be identified through a literature search. For example, genes involved in nitrogen fixation are known in the literature. These known genes can be used as targets for introducing genetic variations. Gene targets can also be identified based on the RNA sequencing data obtained in the step B3 (small-scale field trials for colonization) or by performing RNA sequencing described in the step below.

2. Select Promoters for Promoter Swaps

A desired genetic variation for improving the plant-beneficial activity can comprise promoter swapping, in which the native promoter for a target gene is replaced with a stronger or weaker promoter (when compared to the native promoter) from within the microbe's genome, or differently regulated promoter (e.g. a N-independent). If the expression of a target gene increases the plant-beneficial activity (e.g., nifA, the expression of which enhances nitrogen fixation in microbes), the desired promoter for promoter swapping is a stronger promoter (compared to the native promoter of the target gene) that would further increase the expression level of the target gene compared to the native promoter. If the expression of a target gene decreases the plant-beneficial activity (e.g., nifL that downregulates nitrogen fixation), the desired promoter for promoter swapping is a weak promoter (compared to the native promoter of the target gene) that would substantially decrease the expression level of the target gene compared to the native promoter. Promoters can be inserted into genes to “knock-out” a gene's expression, while at the same time upregulating the expression of a downstream gene.

Promoters for promoter swapping can be selected based on the RNA sequencing data. For example, the RNA sequencing data can be used to identify strong and weak promoters, or constitutively active vs. inducible promoters.

For example, to identify strong and weak promoters, or constitutively active vs. inducible promoters, in the nitrogen fixation pathway, selected microbes will be cultured in vitro under nitrogen-depleted and nitrogen-replete conditions; RNA of the microbe will be isolated from these cultures; and sequenced.

In one protocol, the RNA profile of the microbe under nitrogen-depleted and nitrogen-replete conditions will be compared and active promoters with a desired transcription level will be identified. These promoters can be selected to swap a weak promoter.

Promoters can also be selected using the RNA sequencing data obtained in the step B3 that reflects the RNA profile of the microbe in planta in the host plant rhizosphere.

RNA sequencing under various conditions allows for selection of promoters that: a) are active in the rhizosphere during the host plant growth cycle in fertilized field conditions, and b) are also active in relevant in vitro conditions so they can be rapidly screened.

In an exemplary protocol, in planta RNA sequencing data from colonization assays (e.g. step B3) is used to measure the expression levels of genes in isolated microbes. In one embodiment, the level of gene expression is calculated as reads per kilobase per million mapped reads (RPKM). The expression level of various genes is compared to the expression level of a target gene and at least the top 10, 20, 30, 40, 50, 60, or 70 promoters, associated with the various genes, that show the highest or lowest level of expression compared to the target gene are selected as possible candidates for promoter swapping. Thus, one looks at expression levels of various genes relative to a target gene and then selects genes that demonstrate increased expression relative to a target (or standard) gene and then find the promoters associated with said genes.

For example, if the target gene is upregulation of nifA, the first 10, 20, 30, 40, 50, or 60 promoters for genes that show the highest level of expression compared to nifA are selected as possible candidates for promoter swapping.

These candidates can be further short-listed based on in vitro RNA sequencing data. For example, for nifA as the target gene, possible promoter candidates selected based on the in planta RNA sequencing data are further selected by choosing promoters with similar or increased gene expression levels compared to nifA under in vitro nitrogen-deplete vs. nitrogen-replete conditions.

The set of promoters selected in this step are used to swap the native promoter of the target gene (e.g. nifA). Remodeled strains with swapped promoters are tested in in vitro assays; strains with lower than expected activity are eliminated; and strains with expected or higher than expected activity are tested in field. The cycle of promoter selection may be repeated on remodeled strains to further improve their plant-beneficial activity.

Described here is an exemplary promoter swap experiment that was carried out based on in planta and in vitro RNA sequencing data from Klebsiella variicola strain, CI137 to improve the nitrogen fixation trait. CI137 was analyzed in ARA assays at 0 mM and 5 mM glutamine concentration and RNA was extracted from these ARA samples. The RNA was sequenced via NextSeq and a subset of reads from one sample was mapped to the CI137 genome (in vitro RNA sequencing data). RNA was extracted from the roots of corn plants at V5 stage in the colonization and activity assay (e.g. step B3) for CI137. Samples from 6 plants were pooled; the RNA from the pooled sample was sequenced using NextSeq, and reads were mapped to the CI137 genome (in planta RNA sequencing data). Out of 2×10⁸ total reads, 7×10⁴ reads mapped to CI137. In planta RNA sequencing data was used to rank genes in order of in planta expression levels and the expression levels were compared to the native nifA expression level. The first 40 promoters that showed the highest expression level (based on gene expression) compared to the native nifA expression level were selected. These 40 promoters were further short-listed based on the in vitro RNA sequencing data, where promoters with increased or similar in vitro expression levels compared to nifA were selected. The final list of promoters included 17 promoters and 2 versions of most promoters were used to generate promoter swap mutants; thus a total of 30 promoters were tested. Generation of a suite of CI137 mutants where nifL was deleted partially or completely and the 30 promoters inserted (ΔnifL::Prm) was attempted. 28 out of 30 mutants were generated successfully. The ΔnifL::Prm mutants were analyzed in ARA assays at 0 mM and 5 mM glutamine concentration and RNA was extracted from these ARA samples. Several mutants showed lower than expected or decreased ARA activity compared to the WT CI137 strain. A few mutants showed higher than expected ARA activity.

A person of ordinary skill in the art would appreciate from the above example that while in planta and/or in vitro RNA sequencing data can be used to select promoters for promoter swapping, the step of promoter selection is highly unpredictable and involves many challenges.

For example, in planta RNA sequencing mainly reveals the genes that are highly expressed; however, it is difficult to detect fine differences in gene expression and/or genes with low expression levels. For instance, in some in planta RNA sequencing experiments, only about 40 out of about 5000 genes from a microbial genome were detected. Thus, in planta RNA sequencing technique is useful to identify abundantly expressed genes and their corresponding promoters; however, the technique has difficulty in identifying low expression genes and corresponding promoters and small differences between gene expression.

Furthermore, in planta RNA profile reflects the status of the genes at the time the microbes were isolated; however, a slight change in the field conditions can substantially change the RNA profile of rhizosphere/epiphytic/endophytic microbes. Therefore, it is difficult to predict in advance whether the promoters selected based on one field trial RNA sequencing data would provide desirable expression levels of the target gene when remodeled strains are tested in vitro and in field.

Additionally, in planta evaluation is time and resource-consuming; therefore, in planta experiments cannot be conducted often and/or repeated quickly or easily. On the other hand, while in vitro RNA sequencing can be conducted relatively quickly and easily, the in vitro conditions do not mimic the field conditions and promoters that may show high activity in vitro may not show comparable activity in planta.

Moreover, promoters often don't behave as predicted in a new context. Therefore, in planta and in vitro RNA sequencing data can at best serve as a starting point in the step of promoter selection; however, arriving at any particular promoter that would provide desirable expression levels of the target gene in the field is, in some instances, unpredictable.

Another limitation in the step of promoter selection is the number of available promoters. Because one of the goals of the present invention is to provide non-transgenic microbes; promoters for promoter swapping need to be selected from within the microbe's genome, or genus. Thus, unlike a transgenic approach, the present process can not merely go out into the literature and find/use a well characterized transgenic promoter from a different host organism.

Another constraint is that the promoter must be active in planta during a desired growth phase. For example, the highest requirement for nitrogen in plants is generally late in the growing season, e.g. late vegetative and early reproductive phases. For example, in corn, nitrogen uptake is the highest during V6 (6 leaves) through R1 (reproductive stage 1) stages. Therefore, to increase the availability of nitrogen during V6 through R1 stages of corn, remodeled microbes must show highest nitrogen fixation activity during these stages of the corn lifecycle. Accordingly, promoters that are active in planta during the late vegetative and early reproductive stages of corn need to be selected. This constraint not only reduces the number of promoters that may be tested in promoter swapping, but also make the step of promoter selection unpredictable. As discussed above, unpredictability arises, in part because although the RNA sequencing data from small scale field trials (e.g. step B3) may be used to identify promoters that are active in planta during a desired growth stage, the RNA data is based on the field conditions (e.g., type of soil, level of water in the soil, level of available nitrogen, etc.) at the time of sample collection. As one of ordinary skill in the art would understand, the field conditions may change over the period of time within the same field and also change substantially across various fields. Thus, the promoters selected under one field condition may not behave as expected under other field conditions. Similarly, selected promoters may not behave as expected after swapping. Therefore, it is difficult to anticipate in advance whether the selected promoters would be active in planta during a desired growth phase of a plant of interest

3. Design Non-Intergeneric Genetic Variations

Based on steps D1 (identification of gene targets) and D2 (identification of promoters for promoter swaps), non-intergeneric genetic variations will be designed.

The term “non-intergeneric” indicates that the genetic variation to be introduced into the host does not contain a nucleic acid sequence from outside the host genus (i.e., no transgenic DNA). Although vectors and/or other genetic tools will be used to introduce the genetic variation into the host microbe, the methods of the present disclosure include steps to loop-out (remove) the backbone vector sequences or other genetic tools introduced into the host microbe leaving only the desired genetic variation into the host genome. Thus, the resulting microbe is non-transgenic.

Exemplary non-intergeneric genetic variations include a mutation in the gene of interest that may improve the function of the protein encoded by the gene; a constitutionally active promoter that can replace the endogenous promoter of the gene of interest to increase the expression of the gene; a mutation that will inactivate the gene of interest; the insertion of a promoter from within the host's genome into a heterologous location, e.g. insertion of the promoter into a gene that results in inactivation of said gene and upregulation of a downstream gene; and the like. The mutations can be point mutations, insertions, and/or deletions (full or partial deletion of the gene). For example, in one protocol, to improve the nitrogen fixation activity of the host microbe, a desired genetic variation may comprise an inactivating mutation of the nifL gene (negative regulator of nitrogen fixation pathway) and/or comprise replacing the endogenous promoter of the nifH gene (nitrogenase iron protein that catalyzes a key reaction to fix atmospheric nitrogen) with a constitutionally active promoter that will drive the expression of the nifH gene constitutively.

4. Generate Non-Intergeneric Derivative Strains

After designing the non-intergeneric genetic variations, steps C2-C7 will be carried out to generate non-intergeneric derivative strains (i.e. remodeled microbes).

5. Bank a Purified Culture of the Remodeled Microbe

A purified culture of the remodeled microbe will be preserved in a bank, so that gDNA can be extracted for whole genome sequencing described below.

6. Confirm Presence of the Desired Genetic Variation

The genomic DNA of the remodeled microbe will be extracted and the whole genome sequencing will be performed on the genomic DNA using methods described previously. The resulting reads will be mapped to the reads previously stored in LIMS to confirm: a) presence of the desired genetic variation, and b) complete absence of reads mapping to vector sequences (e.g. plasmid backbone or helper plasmid sequence) that were used to generate the remodeled microbe.

This step allows sensitive detection of non-host genus DNA (transgenic DNA) that may remain in the strain after looping out of the vector backbone (e.g. suicide plasmid) method and could provide a control for accidental off-target insertion of the genetic variation, etc.

E. Analytics Upon Remodeled Microbes

1. Analysis of the Plant-Beneficial Activity

The plant-beneficial activity and growth kinetics of the remodeled microbes will be assessed in vitro.

For example, strains remodeled for improving nitrogen fixation function will be assessed for nitrogen fixation activity and fitness through acetylene reduction assays, ammonium excretion assays, etc.

Strains remodeled for improved phosphate solubilization will be assessed for the phosphate solubilization activity.

This step allows rapid, medium to high throughput screening of remodeled strains for the phenotypes of interest.

2. Analysis of Colonization and Transcription of the Altered Genes

Remodeled strains will be assessed for colonization of the host plant either in the greenhouse or in the field using the steps described in B3. Additionally, RNA will be isolated from colonized root and/or soil samples and sequenced to analyze the transcriptional activity of target genes. Target genes comprise the genes containing the genetic variation introduced and may also comprise other genes that play a role in the plant-beneficial trait of the microbe.

For example, a cluster of genes, the nif genes, controls the nitrogen fixation activity of microbes. Using the protocol described above, a genetic variation may be introduced into one of the nif genes (e.g. a promoter insertion), whereas the other genes in the nif cluster are in their endogenous form (i.e. their gene sequence and/or the promoter region is not altered). The RNA sequencing data will be analyzed for the transcriptional activity of the nif gene containing the genetic variation and may also be analyzed for other nif genes that are not altered directly, by the inserted genetic change, but nonetheless may be influenced by the introduced genetic change.

This step allows determination of the fitness of top in vitro performing strains in the rhizosphere and allows measurement of the transcriptional activity of altered genes in planta.

F. Iterate Engineering Campaign/Analytics

The data from in vitro and in planta analytics (steps E1 and E2) will be used to iteratively stack beneficial mutations.

Furthermore, steps A-E described above may be repeated to fine tune the plant-beneficial traits of the microbes. For example, plants will be inoculated using microbial strains remodeled in the first round; harvested after a few weeks of growth; and microbes from the soil and/or roots of the plants will be isolated. The functional activity (plant-beneficial trait and colonization potential) and the DNA and RNA profile of isolated microbes will be characterized, in order to select microbes with improved plant-beneficial activity and colonization potential. The selected microbes will be remodeled to further improve the plant-beneficial activity. Remodeled microbes will be screened for the functional activity (plant-beneficial trait and colonization potential) and RNA profile in vitro and in planta and the top performing strains will be selected. If desired, steps A-E can be repeated to further improve the plant-beneficial activity of the remodeled microbes from the second round. The process can be repeated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more rounds.

The exemplary steps described above are summarized in Table A below.

TABLE A An Overview of an Embodiment of the Guided Microbial Remodeling Platform Steps Contribution Alternate Forms A Isolation 1 Obtain a soil sample Provides WT soil microbes to be isolated 2 Grow corn “bait Allows selection of plant- Wheat, sorghum, rice, millet, plants” in soil sample beneficial microbes by soybean, etc. rhizosphere 3 Harvest, clean and Down-select soil microbes Other nitrogen-free media, other extract root sample to those that a) colonize the selective or screening media (eg. and plate on nitrogen- root and b) fix atmospheric for phosphate solubilization) free (specifically nitrogen NfB) media 4 Pick colonies, purify Down-select microbes to Degenerate primers for other cultures and screen those containing the nifH genes of interest, e.g. ipdC for presence of nifH gene (eliminate false- (phytohormone biosynthesis) using degenerate positive from media primers screen) 5 Bank a purified culture of the strain B Characterization 1 Sequence and Characterize genome for assemble the genome key pathways of the strain using Illumina and/or PacBio platform 2 Assay the microbe for Down-select for microbes Wheat, sorghum, rice, millet, colonization of corn that colonize the plant well soybean, etc., other methods for roots in the assaying colonization (e.g. greenhouse (qPCR- plating) based method) 3 Assay the microbe for Known internally as “CAT” Larger field trials, other crops, colonization of com trials, these provide other methods for assaying roots in a small-scale Colonization And colonization (e.g. plating) field trials (qPCR- Transcript data for the based method) and strain in a field isolate RNA from environment colonized root samples 4 Assay the microbe for Confirm N-fixation nitrogen fixation phenotype of strain activity in an acetylene reduction assay (ARA) 5 Use the above data to Allows selection of select candidate greatest-potential microbe for further candidates domestication and optimization C Domestication 1 Test microbes for Determine which antibiotic sensitivity to various selection markers can be antibiotics used to transform genetic tools 2 Design and build a These are the “parts” Plasmid could contain a SceI site suicide plasmid necessary to maintain the or other counter-selectable containing an plasmid and carry out marker, alternate fluorescent appropriate antibiotic conjugation, insertion and reporters, additional elements resistance marker, “loop-out” of the host sacB counter- genome selectable marker, origin of replication for maintenance in E. coli, GFP to screen for insertion through fluorescence, origin of transfer for conjugation into the host, homology arms to the host genome, and the desired mutation. 3 Transform suicide Preparation for conjugation Could use a different donor strain plasmid into E. coli into host; plasmid of E. coli or other microbe; ST18 (an auxotroph maintenance different auxotrophic marker for aminolevulinic acid, ALA) to generate donor cells 4 Mix donor cells with The suicide plasmid is able Could use a different donor strain recipient host cells to to replicate in E. coli but of E. coli or other microbe; conjugate, and plate not in the host Therefore different auxotrophic marker on media selecting for plating of the mixture on the antibiotic such plates means that only resistance marker and host cells that received the NOT containing ALA plasmid and experience plasmid integration into the chromosome will be able to grow and form colonies. The E coli ST18 is unable to grow due to the absence of ALA 5 Confirm integration Confirms proper integration of the plasmid of the suicide plasmid through GFP backbone containing GFP, fluorescence, and the antibiotic resistance integration at the cassette, the sacB marker, intended locus etc. through colony PCR 6 Streak confirmed The sacB marker confers Different counter selectable integration colony on sensitivity to sucrose; marker, SceI-mediated loop-out, a plate containing colonies which have etc. sucrose and screen for undergone a second round non-fluorescent of homologous colonies recombination and “looped- out” the plasmid will grow better and not fluoresce on the plate. 7 Screen looped-out Upon the second colonies for the homologous recombination intended mutation event only 50% of looped using colony PCR out colonies should contain the mutation, the other 50% will be WT 8 If any of the steps 2-7 Allows iterative fail, go back to step 2 troubleshooting of suicide and re-design with plasmid to develop a alternate plasmid working protocol parts 9 Once steps 2-7 can be reliably performed, develop an SOP for that strain/plasmid to be used for Optimization D Non-Intergeneric Engineering Campaign and Optimization 1 Identify gene targets for optimizing a pathway, eg. nif genes through literature search 2 Select promoters for Allows for selection of Alternate crops; alternate RNAseq promoter swaps using promoters that a) are active data conditions (greenhouse, field, RNAseq data in the rhizosphere during in vitro, whatever's relevant for collected both in vitro the corn growth cycle in the phenotype targeted) in N-depleted and N- fertilized field conditions b) replete conditions, are also active in in vitro N- and in planta from replete conditions so they the corn rhizosphere can be rapidly screened. (Collected in step B3) 3 Design non- No DNA from outside the Alter regulatory sequences (e.g. intergeneric host chromosome is added, RBS), non-coding RNAs, etc. mutations in key therefore the resulting genes: deletions (full microbe is non-transgenic or partial gene), promoter swaps, or single base pair changes; store these designs in our LIMS 4 Using the established We perform this in higher protocol, carry out throughput than the steps C2-7 to generate domestication step - up to non-intergeneric 20 or so strains at once per derivative strains person. (mutants) 5 Bank a purified culture of the strain, extract gDNA and conduct WGS via Illumina 6 Map the resulting Allows very sensitive Suicide plasmid removal is fairly reads to the designs detection of non- reliable; however use of other stored in LIMS to intergeneric DNA that may stable plasmids in alternate confirm a) presence remain in the strain after methods necessitates this extra of the desire mutation the suicide plasmid method; step to ensure with complete and b) complete confirm absence of confidence that no transgenic absence of reads transgenic DNA, controls DNA that was previously mapping to any for accidental off-target transformed in remains in the suicide plasmid or insertion of the suicide strain. other plasmid plasmid, etc. sequences used to generate the strains E Analytics 1 Analyze the strains Allow rapid, med- to high- Any other in vitro assay, e.g. for in vitro nitrogen throughput screening of phosphate solubilization, qPCR fixation activity and mutants for phenotypes of for transcription of specific genes, fitness through ARA, interest etc. ammonium excretion assays, and growth curves 2 Analyze the strains Measure fitness of top in for colonization vitro performing strains in (qPCR) and the rhizosphere; measure transcription of target transcription of promoter- and promoter- swapped genes in planta swapped genes (Nanostring) in the plant (greenhouse or field) F Iterate Engineering Campaign/Analytics 1 Use data from in vitro and in planta analytics to iteratively stack beneficial mutations.

Traditional Approaches to Creating Biologicals for Agriculture Suffer from Drawbacks Inherent in their Methodology

Unlike pure bioprospecting of wild-type (WT) microbes or transgenic approaches, GMR allows for non-intergeneric genetic optimization of key regulatory networks within the microbe, which improves plant-beneficial phenotypes over WT microbes, but doesn't have the risks associated with transgenic approaches (e.g. unpredictable gene function, public and regulatory concerns). See, FIG. 1C for a depiction of a problematic “traditional bioprospecting” approach, which has several drawbacks compared to the taught GMR platform.

Other methods for developing microbials for agriculture are focused on either extensive lab development, which often fails at the field scale, or extensive greenhouse or “field-first” testing without an understanding of the underlying mechanisms/plant-microbe interactions. See, FIG. 1D for a depiction of a problematic “field-first approach to bioprospecting” system, which has several drawbacks compared to the taught GMR platform.

The GMR Platform Solves these Problems in Numerous Ways

One strength of the GMR platform is the identification of active promoters, which are active at key physiologically important times for a target crop, and which are also active under particular, agriculturally relevant, environmental conditions.

As has been explained, within the context of nitrogen fixation, the GMR platform is able to identify microbial promoter sequences, which are active under environmental conditions of elevated exogenous nitrogen, which thereby allows the remodeled microbe to fix atmospheric nitrogen and deliver it to a target crop plant, under modern agricultural row crop conditions, and at a time when a plant needs the fixed nitrogen the most. See, FIG. 1E for a depiction of the time period in the corn growth cycle, at which nitrogen is needed most by the plant. The taught GMR platform is able to create remodeled microbes that supply nitrogen to a corn plant at the time period in which the nitrogen is needed, and also deliver such nitrogen even in the presence of exogenous nitrogen in the soil environment.

These promoters can be identified by rhizosphere RNA sequencing and read mapping to the microbe's genome sequence, and key pathways can be “reprogrammed” to be turned on or off during key stages of the plant growth cycle. Additionally, through whole genome sequencing of optimized microbes and mapping to previously-transformed sequences, the method has the ability to ensure that no transgenic sequences are accidentally released into the field through off-target insertion of plasmid DNA, low-level retention of plasmids not detected through PCR or antibiotic resistance, etc.

The GMR platform combines these approaches by evaluating microbes iteratively in the lab and plant environment, leading to microbes that are robust in greenhouse and field conditions rather than just in lab conditions.

Various aspects and embodiments of the taught GMR platform can be found in FIGS. 1F-1I. The GMR platform culminates in the derivation/creation/production of remodeled microbes that possess a plant-beneficial property, e.g. nitrogen fixation.

The traditional bioprospecting methods are not able to produce microbes having the aforementioned properties.

Properties of a Microbe Remodeled for Nitrogen Fixation

In the context of remodeling microbes for nitrogen fixation, there are several properties that the remodeled microbe may possess. For instance, FIG. 1J depicts 5 properties that can be possessed by remodeled microbes of the present disclosure.

The present inventors have utilized the GMR platform to produce remodeled non-intergeneric bacteria (i.e. Kosakonia sacchari) capable of fixing atmospheric nitrogen and delivering said nitrogen to a corn plant, even under conditions in which exogenous nitrogen is present in the environment. See, FIG. 1K-M, which illustrate that the remodeling process successfully: (1) decoupled nifA expression from endogenous nitrogen regulation; and (2) improved the assimilation and excretion of fixed nitrogen.

These remodeled microbes ultimately result in corn yield improvement, when applied to corn crops. See, FIG. 1N.

The GMR Platform Provides an Approach to Nitrogen Fixation and Delivery that Solves Pressing Environmental Concerns

As explained previously, the nitrogen fertilizer produced by the industrial Haber-Bosch process is not well utilized by the target crop. Rain, runoff, heat, volatilization, and the soil microbiome degrade the applied chemical fertilizer. This equates to not only wasted money, but also adds to increased pollution instead of harvested yield. To this end, the United Nations has calculated that nearly 80% of fertilizer is lost before a crop can utilize it. Consequently, modern agricultural fertilizer production and delivery is not only deleterious to the environment, but it is extremely inefficient. See, FIG. 1O, illustrating the inefficiency of current nitrogen delivery systems, which result in under fertilized fields, over fertilized fields, and environmentally deleterious nitrogen runoff.

The current GMR platform, and resulting remodeled microbes, provide a better approach to nitrogen fixation and delivery to plants. The non-intergeneric remodeled microbes of the disclosure are able to colonize the roots of a corn plant and spoon feed said corn plants with fixed atmospheric nitrogen, even in the presence of exogenous nitrogen. This system of nitrogen fixation and delivery—enabled by the taught GMR platform—will help transform modern agricultural to a more environmentally sustainable system.

Table 25 and Table 26 describe microbes, their underlying genetic architecture, and their corresponding SEQ ID NOs. These microbes have been derived utilizing the GMR platform described in Example 1.

TABLE 25 WT and Remodeled Non-intergeneric Microbes Strain Name Genotype SEQ ID NO CI006 16S rDNA - contig 5 62 CI006 16S rDNA - contig 8 63 CI019 16S rDNA 64 CI006 nifH 65 CI006 nifD 66 CI006 nifK 67 CI006 nifL 68 CI006 nifA 69 CI019 nifH 70 CI019 nifD 71 CI019 nifK 72 CI019 nifL 73 CI019 nifA 74 CI006 Prm5 with 500 bp 75 flanking regions CI006 nifLA operon - upstream 76 intergenic region plus nifL and nifA CDSs CI006 nifL (Amino Acid) 77 CI006 nifA (Amino Acid) 78 CI006 glnE 79 CI006 glnE_KO1 80 CI006 glnE (Amino Acid) 81 CI006 glnE_KO1 (Amino Acid) 82 CI006 GlnE ATase domain 83 (Amino Acid) CM029 Prm5 inserted into nifL 84 region

TABLE 26 WT and Remodeled Non-intergeneric Microbes Associated Novel Strain SEQ ID Junction If Strain ID NO Genotype Description Applicable CI63; 63 SEQ ID 16S N/A N/A CI063 NO 85 CI63; 63 SEQ ID nifH N/A N/A CI063 NO 86 C163; 63 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A CI063 NO 87 in 63 genome CI63; 63 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A CI063 NO 88 in 63 genome CI63; 63 SEQ ID nifK1 1 of 2 unique genes annotated as nifK N/A CI063 NO 89 in 63 genome CI63; 63 SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A CI063 NO 90 in 63 genome CI63; 63 SEQ ID nifL N/A N/A CI063 NO 91 CI63; 63 SEQ ID nifA N/A N/A CI063 NO 92 CI63; 63 SEQ ID glnE N/A N/A CI063 NO 93 CI63; 63 SEQ ID amtB N/A N/A CI063 NO 94 CI63; 63 SEQ ID PinfC 500 bp immediately upstream of the N/A CI063 NO 95 ATG start codon of the infC gene CI137 137 SEQ ID 16S N/A N/A NO 96 CI137 137 SEQ ID nifH1 1 of 2 unique genes annotated as nifH N/A NO 97 in 137 genome CI137 137 SEQ ID nifH2 2 of 2 unique genes annotated as nifH N/A NO 98 in 137 genome CI137 137 SEQ ID nifD1 I of 2 unique genes annotated as nifD N/A NO 99 in 137 genome CI137 137 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A NO 100 in 137 genome CI137 137 SEQ ID nifK1 I of 2 unique genes annotated as nifK N/A NO 101 in 137 genome CI137 137 SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A NO 102 in 137 genome CI137 137 SEQ ID nifL N/A N/A NO 103 CI137 137 SEQ ID nifA N/A N/A NO 104 CI137 137 SEQ ID glnE N/A N/A NO 105 CI137 137 SEQ ID PinfC 500 bp immediately upstream of the N/A NO 106 TTG start codon of infC CI137 137 SEQ ID amtB N/A N/A NO 107 CI137 137 SEQ ID Prm8.2 internal promoter located in nlpI gene; N/A NO 108 299 bp starting at 81 bp after the A of the ATG of the nlpI gene CI137 137 SEQ ID Prm6.2 300 bp upstream of the secE gene N/A NO 109 starting at 57 bp upstream of the A of the ATG of secE CI137 137 SEQ ID Prm1.2 400 bp immediately upstream of the N/A NO 110 ATG of cspE gene none 728 SEQ ID 16S N/A N/A NO 111 none 728 SEQ ID nifH N/A N/A NO 112 none 728 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A NO 113 in 728 genome none 728 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A NO 114 in 728 genome none 728 SEQ ID nifK1 1 of 2 unique genes annotated as nifK N/A NO 115 in 728 genome none 728 SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A NO 116 in 728 genome none 728 SEQ ID nifL N/A N/A NO 117 none 728 SEQ ID nifA N/A N/A NO 118 none 728 SEQ ID glnE N/A N/A NO 119 none 728 SEQ ID amtB N/A N/A NO 120 none 850 SEQ ID 16S N/A N/A NO 121 none 852 SEQ ID 16S N/A N/A NO 122 none 853 SEQ ID 16S N/A N/A NO 123 none 910 SEQ ID 16S N/A N/A NO 124 none 910 SEQ ID nifH N/A N/A NO 125 none 910 SEQ ID Dinitrogenase iron- N/A N/A NO 126 molybdenum cofactor CDS none 910 SEQ ID nifD1 N/A N/A NO 127 none 910 SEQ ID nifD2 N/A N/A NO 128 none 910 SEQ ID nifK1 N/A N/A NO 129 none 910 SEQ ID nifK2 N/A N/A NO 130 none 910 SEQ ID nifL N/A N/A NO 131 none 910 SEQ ID nifA N/A N/A NO 132 none 910 SEQ ID glnE N/A N/A NO 133 none 910 SEQ ID amtB N/A N/A NO 134 none 910 SEQ ID PinfC 498 bp immediately upstream of the N/A NO 135 ATG of the infC gene none 1021 SEQ ID 16S N/A N/A NO 136 none 1021 SEQ ID nifH N/A N/A NO 137 none 1021 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A NO 138 in 910 genome none 1021 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A NO 139 in 910 genome none 1021 SEQ ID nifK1 1 of 2 unique genes annotated as nifK N/A NO 140 in 910 genome none 1021 SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A NO 141 in 910 genome none 1021 SEQ ID nifL N/A N/A NO 142 none 1021 SEQ ID nifA N/A N/A NO 143 none 1021 SEQ ID glnE N/A N/A NO 144 none 1021 SEQ ID amtB N/A N/A NO 145 none 1021 SEQ ID PinfC 500 bp immediately upstream of the N/A NO 146 ATG start codon of the infC gene none 1021 SEQ ID Prm1 348 bp includes the 319 bp immediately N/A NO 147 upstream of the ATG start codon of the lpp gene and the first 29 bp of the lpp gene none 1021 SEQ ID Prm7 339 bp upstream of the sspA gene, N/A NO 148 ending at 46 bp upstream of the ATG of the sspA gene none 1113 SEQ ID 16S N/A N/A NO 149 none 1113 SEQ ID nifH N/A N/A NO 150 none 1113 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A NO 151 in 1113 genome none 1113 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A NO 152 in 1113 genome none 1113 SEQ ID nifK N/A N/A NO 153 none 1113 SEQ ID nifL N/A N/A NO 154 none 1113 SEQ ID nifA partial gene due to a gap in the sequence assembly, N/A NO 155 we can only identify a partial gene from the 1113 genome none 1113 SEQ ID glnE N/A N/A NO 156 none 1116 SEQ ID 16S N/A NO 157 none 1116 SEQ ID nifH N/A NO 158 none 1116 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A NO 159 in 1116 genome none 1116 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A NO 160 in 1116 genome none 1116 SEQ ID nifK1 1 of 2 unique genes annotated as nifK N/A NO 161 in 1116 genome none 1116 SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A NO 162 in 1116 genome none 1116 SEQ ID nifL N/A N/A NO 163 none 1116 SEQ ID nifA N/A N/A NO 164 none 1116 SEQ ID glnE N/A N/A NO 165 none 1116 SEQ ID amtB N/A N/A NO 166 none 1293 SEQ ID 16S N/A N/A NO 167 none 1293 SEQ ID nifH N/A N/A NO 168 none 1293 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A NO 169 in 1293 genome none 1293 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A NO 170 in 1293 genome none 1293 SEQ ID nifK 1 of 2 unique genes annotated as nifK N/A NO 171 in 1293 genome none 1293 SEQ ID nifK1 2 of 2 unique genes annotated as nifK N/A NO 172 in 1293 genome none 1293 SEQ ID nifA N/A N/A NO 173 none 1293 SEQ ID glnE N/A N/A NO 174 none 1293 SEQ ID amtB1 1 of 2 unique genes annotated as amtB N/A NO 175 in 1293 genome none 1293 SEQ ID amtB2 2 of 2 unique genes annotated as amtB N/A NO 176 in 1293 genome none 1021-1612  SEQ ID ΔnifL::PinfC starting at 24 bp after the A of the ATG ds1131 NO 177 start codon, 1375 bp of nifL have been deleted and replaced with the 1021 PinfC promoter sequence none 1021-1612  SEQ ID ΔnifL::PinfC with starting at 24 bp after the A of the ATG ds1131 NO 178 500 bp flank start codon, 1375 bp of nifL have been deleted and replaced with the 1021 PinfC promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included none 1021-1612  SEQ ID glnEΔAR-2 glnE gene with 1673 bp immediately ds1133 NO 179 downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain none 1021-1612  SEQ ID glnEΔAR-2 with glnE gene with 1673 bp immediately ds1133 NO 180 500 bp flank downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE gene upstream and downstream are included none 1021-1615  SEQ ID ΔnifL::Prm1 starting at 24 bp after the A of the ATG ds1145 NO 181 start codon, 1375 bp of nifL have been deleted and replaced with the 1021 Prm1 promoter sequence none 1021-1615  SEQ ID ΔnifL::Prm1 with starting at 24 bp after the A of the ATG ds1145 NO 182 500 bp flank start codon, 1375 bp of nifL have been deleted and replaced with the 1021 rm1 promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included none 1021-1615  SEQ ID glnEΔAR-2 glnE gene with 1673 bp immediately ds1133 NO 183 downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain none 1021-1615  SEQ ID glnEΔAR-2 with glnE gene with 1673 bp immediately ds1133 NO 184 500 bp flank downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE gene upstream and downstream are included none 1021-1619  SEQ ID ΔnifL::Prm1 starting at 24 bp after the A of the ATG ds1145 NO 185 start codon, 1375 bp of nifL have been deleted and replaced with the 1021 Prm1 promoter sequence none 1021-1619  SEQ ID ΔnifL::Prm1 with starting at 24 bp after the A of the ATG ds1145 NO 186 500 bp flank start codon, 1375 bp of nifL have been deleted and replaced with the 1021 rm1 promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included none 1021-1623  SEQ ID glnEΔAR-2 glnE gene with 1673 bp immediately ds1133 NO 187 downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain none 1021-1623  SEQ ID glnEΔAR-2 with glnE gene with 1673 bp immediately ds1133 NO 188 500 bp flank downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE gene upstream and downstream are included none 1021-1623  SEQ ID ΔnifL::Prm7 starting at 24 bp after the A of the ATG ds1148 NO 189 start codon, 1375 bp of nifL have been deleted and replaced with the 1021 Prm7 promoter sequence none 1021-1623  SEQ ID ΔnifL::Prm7 with starting at 24 bp after the A of the ATG ds1148 NO 190 500 bp flank start codon, 1375 bp of nifL have been deleted and replaced with the 1021 rm7 promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included none 137-1034 SEQ ID glnEΔAR-2 glnE gene with 1290 bp immediately ds809 NO 191 downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain none 137-1034 SEQ ID glnEΔAR-2 with glnE gene with 1290 bp immediately ds809 NO 192 500 bp flank downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE gene upstream and downstream are included none 137-1036 SEQ ID ΔnifL::PinfC starting at 24 bp after the A of the ATG ds799 NO 193 start codon, 1372 bp of nifL have been deleted and replaced with the 137 PinfC promoter sequence none 137-1036 SEQ ID ΔnifL::PinfC with starting at 24 bp after the A of the ATG ds799 NO 194 500 bp flank start codon, 1372 bp of nifL have been deleted and replaced with the 137 PinfC promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included none 137-1314 SEQ ID glnEΔAR-2 36 bp glnE gene with 1290 bp immediately none NO 195 deletion downstream of the ATG start codon deleted AND 36 bp deleted beginning at 1472 bp downstream of the start codon, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain none 137-1314 SEQ ID glnEΔAR-2 36 bp glnE gene with 1290 bp immediately none NO 196 deletion downstream of the ATG start codon deleted AND 36 bp deleted beginning at 1472 bp downstream of the start codon, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain; 500 bp flanking the nifL gene upstream and downstream are included none 137-1314 SEQ ID ΔnifL::Prm8.2 starting at 24 bp after the A of the ATG ds857 NO 197 start codon, 1372 bp of nifL have been deleted and replaced with the 137 Prm8.2 promoter sequence none 137-1314 SEQ ID ΔnifL::Prm8.2 with starting at 24 bp after the A of the ATG ds857 NO 198 500 bp flank start codon, 1372 bp of nifL have been deleted and replaced with the 137 Prm8.2 promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included none 137-1329 SEQ ID glnEΔAR-2 36 bp glnE gene with 1290 bp immediately none NO 199 deletion downstream of the ATG start codon deleted AND 36 bp deleted beginning at 1472 bp downstream of the start codon, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain none 137-1329 SEQ ID glnEΔAR-2 36 bp glnE gene with 1290 bp immediately none NO 200 deletion downstream of the ATG start codon deleted AND 36 bp deleted beginning at 1472 bp downstream of the start codon, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain; 500 bp flanking the nifL gene upstream and downstream are included none 137-1329 SEQ ID ΔnifL::Prm6.2 starting at 24 bp after the A of the ATG ds853 NO 201 start codon, 1372 bp of nifL, have been deleted and replaced with the 137 Prm6.2 promoter sequence none 137-1329 SEQ ID ΔmfL::Prm6.2 with starting at 24 bp after the A of the ATG ds853 NO 202 500 bp flank start codon, 1372 bp of nifL have been deleted and replaced with the 137 Prm6.2 promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included none 137-1382 SEQ ID ΔnifL::Prm1.2 starting at 24 bp after the A of the ATG ds843 NO 203 start codon, 1372 bp of nifL have been deleted and replaced with the 137 Prm1.2 promoter sequence none 137-1382 SEQ ID ΔnifL::Prm1.2 with starting at 24 bp after the A of the ATG ds843 NO 204 500 bp flank start codon, 1372 bp of nifL have been deleted and replaced with the 137 Prm1.2 promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included none 137-1382 SEQ ID glnEΔAR-2 36 bp glnE gene with 1290 bp immediately none NO 205 deletion downstream of the ATG start codon deleted AND 36 bp deleted beginning at 1472 bp downstream of the start codon, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain none 137-1382 SEQ ID glnEΔAR-2 36 bp glnE gene with 1290 bp immediately none NO 206 deletion downstream of the ATG start codon deleted AND 36 bp deleted beginning at 1472 bp downstream of the start codon, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain; 500 bp flanking the nifL gene upstream and downstream are included none 137-1586 SEQ ID ΔnifL::PinfC starting at 24 bp after the A of the ATG ds799 NO 207 start codon, 1372 bp of nifL have been deleted and replaced with the 137 PinfC promoter sequence none 137-1586 SEQ ID ΔnifL::PinfC with starting at 24 bp after the A of the ATG ds799 NO 208 500 bp flank start codon, 1372 bp of nifL have been deleted and replaced with the 137 PinfC promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included none 137-1586 SEQ ID glnEΔAR-2 glnE gene w ith 1290 bp immediately ds809 NO 209 downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain none 137-1586 SEQ ID glnEΔAR-2 with glnE gene with 1290 bp immediately ds809 NO 210 500 bp flank downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE gene upstream and downstream are included none 19-594 SEQ ID glnEΔAR-2 glnE gene with 1650 bp immediately ds34 NO 211 downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain none 19-594 SEQ ID glnEΔAR-2 with glnE gene with 1650 bp immediately ds34 NO 212 500 bp flank downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE gene upstream and downstream are included none 19-594 SEQ ID ΔnifL::Prm6.1 starting at 221 bp after the A of the ds180 NO 213 ATG start codon, 845 bp of nifL have been deleted and replaced with the CI019 Prm6.1 promoter sequence none 19-594 SEQ ID ΔnifL::Prm6.1 with starting at 221 bp after the A of the ds180 NO 214 500 bp flank ATG start codon, 845 bp of nifL have been deleted and replaced with the CI019 Prm6.1promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included none 19-714 SEQ ID ΔnifL::Prm6.1 starting at 221 bp after the A of the ds180 NO 215 ATG start codon, 845 bp of nifL have been deleted and replaced with the CI019 Prm6.1 promoter sequence none 19-714 SEQ ID ΔnifL::Prm6.1 with starting at 221 bp after the A of the ds180 NO 216 500 bp flank ATG start codon, 845 bp of nifL have been deleted and replaced with the CI019 Prm6.1promotor sequence; 500 bp flanking the nifL gene upstream and downstream are included none 19-715 SEQ ID ΔnifL::Prm7.1 starting at 221 bp after the A of the ds181 NO 217 ATG start codon, 845 bp of nifL have been deleted and replaced with the CI019 Prm7.1 promoter sequence none 19-715 SEQ ID ΔnifL::Prm7.1 with starting at 221 bp after the A of the ds181 NO 218 500 bp flank ATG start codon, 845 bp of nifL have been deleted and replaced with the CI019 Prm76.1promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included 19-713 19-750 SEQ ID ΔnifL::Prm1.2 starting at 221 bp after the A of the ds172 NO 219 ATG start codon, 845 bp of nifL have been deleted and replaced with the CI019 Prm1.2 promoter sequence 19-713 19-750 SEQ ID ΔnifL::Prm1.2 with starting at 221 bp after the A of the ds172 NO 220 500 bp flank ATG start codon, 845 bp of nifL have been deleted and replaced with the CI019 Prm1.2 promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included 17-724 19-804 SEQ ID ΔnifL::Prm1.2 starting at 221 bp after the A of the ds172 NO 221 ATG start codon, 845 bp of nifL have been deleted and replaced with the CI019 Prm1.2 promoter sequence 17-724 19-804 SEQ ID ΔnifL::Prm1.2 with starting at 221 bp after the A of the ds172 NO 222 500 bp flank ATG start codon, 845 bp of nifL have been deleted and replaced with the CI019 Prm1.2 promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included 17-724 19-804 SEQ ID glnEΔAR-2 glnE gene with 1650 bp immediately ds34 NO 223 downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain 17-724 19-804 SEQ ID glnEΔAR-2 with glnE gene with 1650 bp immediately ds34 NO 224 500 bp flank downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE gene upstream and downstream are included 19-590 19-806 SEQ ID ΔnifL::Prm3.1 starting at 221 bp after the A of the ds!75 NO 225 ATG start codon, 845 bp of nifL have been deleted and replaced with the CI019 Prm3.1 promoter sequence 19-590 19-806 SEQ ID ΔnifL::Prm3.1 with starting at 221 bp after the A of the dsl75 NO 226 500 bp flank ATG start codon, 845 bp of nifL have been deleted and replaced with the CI019 Prm3.1 promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included 19-590 19-806 SEQ ID glnEΔAR-2 glnE gene with 1650 bp immediately ds34 NO 227 downstream of the ATG s tart codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain 19-590 19-806 SEQ ID glnEΔAR-2 with glnE gene with 1650 bp immediately ds34 NO 228 500 bp flank downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE gene upstream and downstream are included none  63-1146 SEQ ID ΔnifL::PinfC starting at 24 bp after the A of the ATG ds908 NO 229 start codon, 1375 bp of nifL have been deleted and replaced with the 63 PinfC promoter sequence none  63-1146 SEQ ID ΔnifL::PinfC with starting at 24 bp after the A of the ATG ds908 NO 230 500 bp flank start codon, 1375 bp of nifL have been deleted and replaced with the 63 PinfC promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included CM015;  6-397 SEQ ID ΔnifL::Prm5 starting at 31 bp after the A of the ATG ds24 PBC6.15 NO 231 start codon, 1375 bp of nifL have been deleted and replaced with the CI006 Prm5 promoter sequence CM015;  6-397 SEQ ID ΔnifL::Prm5 with starting at 31 bp after the A of the ATG ds24 PBC6.15 NO 232 500 bp flank start codon, 1375 bp of nifL have been deleted and replaced with the CI006 Prm5 promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included CM014  6-400 SEQ ID ΔnifL::Prm1 starting at 31 bp after the A of the ATG ds20 NO 233 start codon, 1375 bp of nifL have been deleted and replaced with the CI006 Prm1 promoter sequence CM014  6-400 SEQ ID ΔnifL::Prm1 with starting at 31 bp after the A of the ATG ds20 NO 234 500 bp flank start codon, 1375 bp of nifL have been deleted and replaced with the CI006 Prm1 promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included CM037;  6-403 SEQ ID ΔnifL::Prm1 starting at 31 bp after the A of the ATG ds20 PBC6.37 NO 235 start codon, 1375 bp of nifL have been deleted and replaced with the CI006 Prm1 promoter sequence CM037;  6-403 SEQ ID ΔnifL::Prm1 with starting at 31 bp after the A of the ATG ds20 PBC6.38 NO 236 500 bp flank start codon, 1375 bp of nifL have been deleted and replaced with the CI006 Pmil promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included CM037;  6-403 SEQ ID glnEΔAR-2 glnE gene with 1644 bp immediately ds31 PBC6.39 NO 237 downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain CM037;  6-403 SEQ ID glnEΔAR-2 with glnE gene with 1644 bp immediately ds31 PBC6.40 NO 238 500 bp flank downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE gene upstream and downstream are included CM038;  6-404 SEQ ID glnEΔAR-1 glnE gene with 1287 bp immediately ds30 PBC6.38 NO 239 downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain CM038;  6-404 SEQ ID ΔnifL::Prm1 starting at 31 bp after the A of the ATG ds20 PBC6.38 NO 240 start codon, 1375 bp of nifL have been deleted and replaced with the CI006 Prm1 promoter sequence CM038;  6-404 SEQ ID ΔnifL::Prm1 with starting at 31 bp after the A of the ATG ds20 PBC6.38 NO 241 500 bp flank start codon, 1375 bp of nifL have been deleted and replaced with the CI006 Prm1 promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included CM038;  6-404 SEQ ID glnEΔAR-1 with glnE gene with 1287 bp immediately ds30 PBC6.38 NO 242 500 bp flank downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE gene upstream and downstream are included CM029;  6-412 SEQ ID glnEΔAR-1 glnE gene with 1287 bp immediately ds30 PBC6.29 NO 243 downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain CM029;  6-412 SEQ ID glnEΔAR-1 with glnE gene with 1287 bp immediately ds30 PBC6.29 NO 244 500 bp flank downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE gene upstream and downstream are included CM029;  6-412 SEQ ID ΔnifL::Prm5 starting at 31 bp after the A of the ATG ds24 PBC6.29 NO 245 start codon, 1375 bp of nifL have been deleted and replaced with the CI006 Prm5 promoter sequence CM029;  6-412 SEQ ID ΔnifL::Prm5 with starting at 31 bp after the A of the ATG ds24 PBC6.29 NO 246 500 bp flank start codon, 1375 bp of nifL have been deleted and replaced with the CI006 Prm5 promoter sequence; 500 bp flanking the nifL gene upstream and dowmstream are included CM093;  6-848 SEQ ID ΔnifL::Prm1 starting at 31 bp after the A of the ATG ds20 PBC6.93 NO 247 start codon, 1375 bp of nifL have been deleted and replaced with the CI006 Prm1 promoter sequence CM093;  6-848 SEQ ID ΔnifL::Prm1 with starting at 31 bp after the A of the ATG ds20 PBC6.93 NO 248 500 bp flank start codon, 1375 bp of nifL have been deleted and replaced with the CI006 Prm1 promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included CM093;  6-848 SEQ ID glnEΔAR-2 glnE gene with 1644 bp immediately ds31 PBC6.93 NO 249 downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain CM093;  6-848 SEQ ID glnEΔAR-2 with glnE gene with 1644 bp immediately ds31 PBC6.93 NO 250 500 bp flank downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE gene upstream and downstream are included CM093;  6-848 SEQ ID ΔamtB First 1088 bp of amtB gene and 4 bp ds126 PBC6.93 NO 251 upstream of start codon deleted; 199 bp of gene remaining lacks a start codon; no amtB protein is translated CM093;  6-848 SEQ ID ΔamtB with 500 bp First 1088 bp of amtB gene and 4 bp ds126 PBC6.93 NO 252 flank upstream of start codon deleted; 199 bp of gene remaining lacks a start codon; no amtB protein is translated CM094;  6-881 SEQ ID glnEΔAR-1 glnE gene w ith 1287 bp immediately ds30 PBC6.94 NO 253 downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain CM094;  6-881 SEQ ID glnEΔAR-1 with glnE gene with 1287 bp immediately ds30 PBC6.94 NO 254 500 bp flank downstream of the ATG start codon deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE gene upstream and downstream are included CM094;  6-881 SEQ ID ΔnifL:Prm1 starting at 31 bp after the A of the ATG ds20 PBC6.94 NO 255 start codon, 1375 bp of nifL have been deleted and replaced with the CI006 Prm1 promoter sequence CM094;  6-881 SEQ ID ΔnifL::Prm1 with starting at 31 bp after the A of the ATG ds20 PBC6.94 NO 256 500 bp flank start codon, 1375 bp of nifL have been deleted and replaced with the CI006 Prm1 promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included CM094;  6-881 SEQ ID ΔamtB First 1088 bp of amtB gene and 4 bp ds126 PBC6.94 NO 257 upstream of start codon deleted; 199 bp of gene remaining lacks a start codon; no amtB protein is translated CM094;  6-881 SEQ ID ΔamtB with 500 bp First 1088 bp of amtB gene and 4 bp ds126 PBC6.94 NO 258 flank upstream of start codon deleted; 199 bp of gene remaining lacks a start codon; no amtB protein is translated none 910-1246 SEQ ID ΔnifL::PinfC starting at 20 bp after the A of the ATG ds960 NO 259 start codon, 1379 bp of nifL have been deleted and replaced with the 910 PinfC promoter sequence none 910-1246 SEQ ID ΔnifL::PinfC with starting at 20 bp after the A of the ATG ds960 NO 260 500 bp flank start codon, 1379 bp of nifL have been deleted and replaced with the 910 PinfC promoter sequence; 500 bp flanking the nifL gene upstream and downstream are included PBC6.1, CI006 SEQ ID 16S-1 1 of 3 unique 16S rDNA genes in the N/A 6, CI6 NO 261 CI006 genome PBC6.1, CI006 SEQ ID 16S-2 2 of 3 unique 16S rDNA genes in the N/A 6, CI6 NO 262 CI006 genome PBC6.1, CI006 SEQ ID nifH N/A N/A 6, CI6 NO 263 PBC6.1, CI006 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A 6, CI6 NO 264 in CI006 genome PBC6.1, CI006 SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A 6. CI6 NO 265 in CI006 genome PBC6.1, CI006 SEQ ID nifL N/A N/A 6. CI6 NO 266 PBC6.1, CI006 SEQ ID nifA N/A N/A 6, CI6 NO 267 PBC6.1, CI006 SEQ ID glnE N/A N/A 6, CI6 NO 268 PBC6.1, CI006 SEQ ID 16S-3 3 of 3 unique 16S rDNA genes in the N/A 6, CI6 NO 269 CI006 genome PBC6.1, CI006 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A 6, CI6 NO 270 in CI006 genome PBC6.1, CI006 SEQ ID nifK1 1 of 2 unique genes annotated as nifK N/A 6, CI6 NO 271 in CI006 genome PBC6.1, CI006 SEQ ID amtB N/A N/A 6, CI6 NO 272 PBC6.1, CI006 SEQ ID Prm1 348 bp includes the 319 bp immediately N/A 6, CI6 NO 273 upstream of the ATG start codon of the lpp gene and the first 29 bp of the lpp gene PBC6.1, CI006 SEQ ID Prm5 313 bp starting at 432 bp upstream of the N/A 6, CI6 NO 274 ATG start codon of the ompX gene and ending 119 bp upstream of the ATG start codon of the ompX gene 19, CI19 CI019 SEQ ID nifL N/A N/A NO 275 19, CI19 CI019 SEQ ID nifA N/A N/A NO 276 19, CI19 CI019 SEQ ID 16S-1 1 of 7 unique 16S rDNA genes in the N/A NO 277 CI019 genome 19, CI19 CI019 SEQ ID 16S-2 2 of 7 unique 16S rDN A genes in the N/A NO 278 CI019 genome 19, CI19 CI019 SEQ ID 16S-3 3 of 7 unique 16S rDNA genes in the N/A NO 279 CI019 genome 19, CI19 CI019 SEQ ID 16S-4 4 of 7 unique 16S rDNA genes in the N/A NO 280 CI019 genome 19, CI19 CT019 SEQ ID 16S-5 5 of 7 unique 16S rDNA genes in the N/A NO 281 CI019 genome 19, CI19 CI019 SEQ ID 16S-6 6 of 7 unique 16S rDNA genes in the N/A NO 282 CI019 genome 19, CI19 CI019 SEQ ID 16S-7 7 of 7 unique 16S rDNA genes in the N/A NO 283 CI019 genome 19, CI19 CI019 SEQ ID nifH1 1 of 2 unique genes annotated as nifH N/A NO 284 in CI019 geno me 19, CI19 CI019 SEQ ID nifH2 2 of 2 unique genes annotated as nifH N/A NO 285 in CI019 genome 19, CI19 CI019 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A NO 286 in CI019 genome 19, CI19 CI019 SEQ ID ntfD2 2 of 2 unique genes annotated as nifD N/A NO 287 in CI019 genome 19, CI19 CI019 SEQ ID nifK1 1 of 2 unique genes annotated as nifK N/A NO 288 in CI019 genome 19, CI19 CI019 SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A NO 289 in CI019 genome 19, CI19 CI019 SEQ ID glnE N/A N/A NO 290 19, C119 CI019 SEQ ID Prm4 449 bp immediately upstream of the N/A NO 291 ATG of the dscC 2 gene 19, CI19 CI019 SEQ ID Prm1.2 500 bp immediately upstream of the N/A NO 292 TTG start codon of the infC gene 19, CI19 CI019 SEQ ID Prm3.1 170 bp immediately upstream of the N/A NO 293 ATG start codon of the rplN gene 19, CI20 CI020 SEQ ID Prm6.1 142 bp immediately upstream of the N/A NO 294 ATG of a highly-expressed hypothetical protein (annotated as PROKKA_00662 in CI019 assembly 82) 19, CI21 CI021 SEQ ID Prm7.1 293 bp immediately upstream of the N/A NO 295 ATG of the lpp gene 19-375, CM67 SEQ ID glnEΔAR-2 glnE gene with 1650 bp immediately ds34 19-417, NO 296 downstream of the ATG start codon CM067 deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain 19-375, CM67 SEQ ID glnEΔAR-2 with glnE gene with 1650 bp immediately ds34 19-417, NO 297 500 bp flank downstream of the ATG start codon CM067 deleted, resulting in a truncated glnE protein lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE gene upstream and downstream are included 19-375, CM67 SEQ ID ΔnifL::null-v1 starting at 221 bp after the A of the none 19-417, NO 298 ATG start codon, 845 bp of nifL have CM067 been deleted and replaced with the 31 bp sequence “GGAGTCTGAACTCATCCTGCGA TGGGGGCTG” 19-375, CM67 SEQ ID ΔnifL::null-v1 with starting at 221 bp after the A of the none 19-417, NO 299 500 bp flank ATG start codon, 845 bp of nifL have CM067 been deleted and replaced with the 31 bp sequence “GGAGTCTGAACTCATCCTGCGA TGGGGGCTG”; 500 bp flanking the nifL gene upstream and downstream are included 19-377, CM69 SEQ ID ΔnifL::null-v2 starting at 221 bp after the A of the none CM069 NO 300 ATG start codon, 845 bp of nifL have been deleted and replaced with the 5 bp sequence “TTAAA” 19-377, CM69 SEQ ID ΔnifL::null-v2 with starting at 221 bp after the A of the none CM069 NO 301 500 bp flank ATG start codon, 845 bp of nifL have been deleted and replaced with the 5 bp sequence “TTAAA”; 500 bp flanking the nifL gene upstream and downstream are included 19-389, CM81 SEQ ID ΔnifL::Prm4 starting at 221 bp after the A of the ds70 19-418, NO 302 ATG start codon, 845 bp of nifL have CM081 been deleted and replaced with the CI19 Prm4 sequence 19-389, CM81 SEQ ID ΔnifL::Prm4 with starting at 221 bp after the A of the ds70 19-418, NO 303 500 bp flank ATG start codon, 845 bp of nifL have CM081 been deleted and replaced with the C119 Prm4 sequence; 500 bp flanking the nifL gene upstream and downstream are included

TABLE 27 Microbial Detection SEQ ID NO SEQ ID NO SEQ ID NO (Junction sequence (comprising (comprising comprising 100 bp up/down 100 bp 100 bp upstream and 100 bp F R base Junction stream upstream of downstream of downstream of Junction primer primer Probe CI Name junction junction) junction) junction) Description SEQ SEQ SEQ 1021 ds1131 up 304 338 372 disrupted N/A N/A N/A nifL gene/ PinfC 1021 ds1131 down 305 339 373 PinfC/ N/A N/A N/A disrupted nifL gene 1021 ds1133 N/A 306 340 374 5′ UTR and N/A N/A N/A ATG/ truncated glnE gene 1021 ds1145 up 307 341 375 disrupted N/A N/A N/A nifL gene/ Prm1 1021 ds1145 down 308 342 376 Prm1/ N/A N/A N/A disrupted nifL gene 1021 ds1148 up 309 343 377 disrupted N/A N/A N/A nifL gene/ Prm7 1021 ds1148 down 310 344 378 Prm4/ N/A N/A N/A disrupted nifL gene CI006 ds126 N/A 311 345 379 5′ UTR up to N/A N/A N/A ATG-4 bp of amtB gene/ disrupted amtB gene CI019 ds172 down 312 346 380 Prm1.2/ SEQ SEQ N/A disrupted ID ID nifL gene NO: NO: 406 C 407 CI019 ds172 up 313 347 381 disrupted N/A N/A N/A nifL gene/ Prm1.2 CI019 ds175 down 314 348 382 Prm3.1/ SEQ SEQ SEQ disrupted ID ID ID nifL gene NO: NO: NO: 408 409 410 CI019 ds175 up 315 349 383 disrupted N/A N/A N/A niL gene/ Prm3.1 CI006 ds20 down 316 350 384 Prm1/ SEQ SEQ SEQ disrupted ID ID ID nifL gene NO: NO: NO: 411 412 413 CI006 ds20 up 317 351 385 disrupted N/A N/A N/A nifL gene/ Prm1 CI006 ds24 up 318 352 386 disrupted SEQ SEQ SEQ nifL gene/ ID ID ID Prm5 NO: NO: NO: 414 415 416 CI006 ds24 down 319 353 387 Prm5/ N/A N/A N/A disrupted nifL gene CI006 ds30 N/A 320 354 388 5′ UTR and N/A N/A N/A ATG/ truncated glnE gene CI006 ds31 N/A 321 355 389 5′ UTR and N/A N/A N/A ATG/ truncated glnE gene CI019 ds34 N/A 32.2 356 390 5′ UTR and N/A N/A N/A ATG/ truncated glnE gene CI019 ds70 up 323 357 391 disrupted N/A N/A N/A nifL gene/ Prm4 CI019 ds70 down 324 358 392 Prm4/ N/A N/A N/A disrupted nifL gene  137 ds799 down 325 359 393 PinfC/ SEQ SEQ SEQ disrupted ID ID ID nifL gene NO: NO: NO: 417 418 419  137 ds799 up 326 360 394 disrupted N/A N/A N/A nifL gene/ PinfC  137 ds809 N/A 327 361 395 5′ UTR and SEQ SEQ SEQ ATG/ ID ID ID truncated NO: NO: NO: glnE gene 420 421 422  137 ds843 up 328 362 396 disrupted N/A N/A N/A nifL gene/ Prm1.2  137 ds843 down 329 363 397 Prm1.2/ N/A N/A N/A disrupted nifL gene  137 ds853 up 330 364 398 disrupted N/A N/A N/A nifL gene/ Prm6.2  137 ds853 down 331 365 399 Prm6.2/ N/A N/A N/A disrupted nifL gene  137 ds857 up 332 366 400 disrupted N/A N/A N/A nifL gene/ Prm8.2  137 ds857 down 333 367 401 Prm8.2/ N/A N/A N/A disrupted nifL gene  63 ds908 down 334 368 402 PinfC/ SEQ SEQ N/A disrupted ID ID nifL gene NO: NO: 423 424  63 ds908 up 335 369 403 disrupted N/A N/A N/A nifL gene/ PinfC  910 ds960 up 336 370 404 disrupted N/A N/A N/A nifL gene/ PinfC  910 ds960 down 337 371 405 PinfC/ N/A N/A N/A disrupted nifL gene

Example 2: Plant Colonization Assays Utilizing Natural Microbial Barcodes to Determine Relative Abundance Objective

The following example is a proof of concept of plant colonization assays that can utilize natural microbial barcodes in order to improve upon current plant colonization assays. In particular, the assays described in this example can form a part of a holistic Guided Microbial Remodeling (GMR) system, as illustrated in Example 1. As can be seen from the current example, the colonization assays utilizing natural microbial barcodes can offer advantages over assays that rely upon absolute abundance numbers, and therefore offer improvements and/or alternatives upon the system in Example 1.

Background

To recap, the GMR pipeline of Example 1 measures absolute plant-root colonization. Absolute colonization information is a component of the final model, where benefits supplied to a plant are determined by multiplying the count, or absolute colonization number, of microbes by their ability to supply nitrogen to plants. An overview of this workflow can be found, inter alia, in FIG. 2A.

While critical for building a model, absolute colonization quantification is a time and labor intensive process.

The assays described in this example, rather than absolute colonization numbers, utilize relative abundance numbers for colonization profiling. The methodology makes use of DNA sequences (i.e. barcodes) to track differences in microbial abundance at high resolution in various populations, including on plant roots.

These relative abundance determination techniques require less processing and/or offer higher resolution microbial abundance information than absolute colonization methods, in some embodiments.

The overall methodology of this approach is explained in FIG. 3A-D and described further below and throughout this disclosure. In contrast to the absolute colonization methods described above, the process proposed in FIG. 3A-D departs from the GMR protocol of Example 1, because it proposes ranking strains by relative performance in high-resolution competitive assays, in a small number of plants, rather than through lower resolution absolute colonization methods performed in hundreds of plants. As shown in FIG. 3A-B, the general method entails identifying genomic nucleic acid sequences (i.e., at least three) that naturally occur in the bacterium of interest. The first step entails isolating and inserting the identified sequences into a native barcode polynucleotide cassette such that at least one of the sequences act as a barcode, and the two sequences flanking the barcode act as a forward primer binding site and a reverse primer binding site. The next step entails generating the cassette (e.g., by PCR amplifying the cassette utilizing oligonucleotides specific to the forward and reverse primer binding sites in order to generate an amplicon comprising the barcode or through DNA synthesis). The subsequent step entails exposing the bacterium of interest to the generated cassette in order to facilitate insertion of the cassette into the genome of the bacterium of interest, but only if the genome of the bacterium naturally comprises the primer binding sites and the barcode (see FIG. 3C). The primer binding sites and the barcode do not have to occur in close proximity to one another in their native locations in the bacterial genome, nor do they need to naturally occur in a particular orientation. In order to effectively determine the abundance (e.g., relative abundance) of the microbes, each microbe of interest (differentiated by species, strain, or variant) must contain a native barcode polynucleotide cassette that contains a barcode unique to that species, strain, or variant. As shown in FIG. 3D, the next steps in the general method for the relative abundance colonization assay described herein is inoculating a plant of interest with a bacterium containing the native barcode polynucleotide cassette; isolating gDNA from a desired part of the plant (e.g., roots) after it has grown for a desired period of time; amplifying the barcode cassette using primers directed to the forward and reverse primer binding sites present in the barcode cassette; performing sequencing (e.g., next-generation sequencing (NGS)); and estimating the relative abundance of the barcode containing microbe on the plant by determining the relative abundance of sequences of the barcode cassette from the NGS data. In particular, the relative abundance can be determined using the custom analysis pipeline as described herein. In an alternative approach, the gDNA isolated from the plant inoculated with the barcode containing microbe can be used for qPCR analysis with the barcodes serving as probe targets. The relative abundance values determined for a particular isolate can then serve as an internal colonization ladder to which new isolates can be compared. This can be useful in order to determine or detect colonization defects of particular species, strains or variants. The relative abundance colonization assay described herein which comprises amplifying and sequencing of microbial nucleic acid in order to ascertain the colonization capacity of said microbial strains can be referred to as competitive colonization sequencing or Coco-Seq.

Methods and Results

In order to illustrate the concepts shown in FIG. 3A-D, 4 native barcode polynucleotide cassettes were generated for insertion into microbial strains (i.e., parental 137 strain and 137-1036 strain). The barcode sequences were generated through a custom Python script which randomly identified two discrete 24 nucleotide sequences from the genome of the strain of interest and linked them together. The resulting 48 nucleotide sequence, which is considered to be the barcode portion of the native barcode polynucleotide cassette, was screened to avoid start codons, nucleic acid secondary structure, particular enzymatic restriction sites, unbalanced GC content, and low basepair diversity. Only novel barcodes which passed all filters were considered. Three of the native barcode polynucleotides cassettes (designated 137 barcodes 1, 2 and 7) were designed to be unique to strain 137 (parental strain), while the 4^(th) cassette was designed to be unique to the variant strain 137-1036 (i.e., 137-1036 barcode). Each of the native barcode polynucleotide cassettes further contained a 338R forward primer binding site and an 806R reverse primer binding site that flanked the respective barcode sequence. The barcode sequence in each of the 4 cassettes was distinct from each of the other barcodes used in this example.

The 48 base pair barcode sequences utilized in this example were:

137 Barcode_1: (SEQ ID NO: 425) TATCCGTAAGCGAGCAAAGCGTGACGACTGGTCGCGTGACCTGAATGC 137 Barcode_2: (SEQ ID NO: 426) TATCCGTAAGCGAGCAAAGCGTGAGCCATTATGCAGTCGATCGTTCAC 137 Barcode_7: (SEQ ID NO: 427) TATCCGTAAGCGAGCAAAGCGTGAGTGAAGTCTACTACGCTCTCTCAG 137-1036 Barcode: (SEQ ID NO: 428) TATCCGTAAGCGAGCAAAGCGTGAAACGGCACGTCCGAATCGTATCAA

It should be noted that the first 24 basepairs of each of the above barcodes are the same for all of the strains, while the last 24 basepairs vary. This is due to the fact that all of the strains are 137 variants or mutants. Therefore the barcodes were designed such that the first 24 base pairs designate it for a particular lineage (e.g., CI137, CI910 or CI6) while the last 24 base pairs identify a particular variant or mutant (e.g., 137-1036 or, in this case, the parent strain of 137 again).

The 338R and 806R primer binding sites were chosen following testing of a multitude of well-characterized, conserved primer binding site sequences already identified in the 16S rRNA gene (see FIG. 3A). The sequences of each of these primer binding sites (or complements thereof) can be found in Table B.

TABLE B  Primer Names and sequences for use as primer binding sites Pivot LIMS ID Primer Name Sequence 6302 27F AGAGTTTGATCCTGGCTCAG (SEQ ID NO: 429 6303 338R TGCTGCCTCCCGTAGGAGT (SEQ ID NO: 430) 6304 *515F GTGCCAGCMGCCGCGGTAA (SEQ ID NO: 431) 6305 *806R GGACTACHVGGGTWTCTAAT (SEQ ID NO: 432) 6306 *804F AGATTAGATACCCDRGTAGTC (SEQ ID NO: 433) 6307 *1392R ACGGGCGGTGTGTRC (SEQ ID NO: 434) *The 515F, 806R, 804F and 1392R primers comprise mixed bases that follow the mixed-base code for DNA found at www.idtdna.com/pages/products/custom-dna-rna/mixed-bases. In summary, within the primer sequences in Table B, M can be A or C; H can be A, C or T/U; V can be A, C or G; D can be A, G, or T/U; R can be A or G; W can be A or T.

Genomic DNA was isolated from several strains (i.e., CI1293, 63-1146, 1021-1612, 1251, 6, 910 and 137-1036), pooled and subjected to PCR using Phusion polymerase as shown in Table C.

TABLE C PCR protocol for experiment shown in FIG. 4. Step No. of Cycles Temp (Celsius) Time Initial Denaturation 1 95 3 minutes Melt 25 95 20 seconds Anneal 59 45 seconds Extension 72 30 seconds

The sets were paired such that 2 forward and 2 reverse primers were matched and, thus, no product was expected to be produced. As shown in FIG. 4, pairs #1 and #3 had unexpectedly strong amplification, while pairs #2, #5 and #6 had light background amplification. Pair #4 had the predicted result which showed no significant background amplification and was thus chosen as the universal binding sites for the cassettes (i.e., they flank the barcode sequences as described above) used in the colonization assays described in this example.

Each of the native barcode polynucleotide cassettes provided above were synthesized by IDT. It should be noted that during synthesis of the native barcode polynucleotide cassettes, homology arms that comprised sequence complementary to a desired locus or site in the genome of the tested strains were introduced.

The native barcode polynucleotide cassettes generated for the relative abundance studies in this example were each subsequently cloned into a vector (i.e., suicide vector) and transformed into either the parent 137 strain (i.e., barcodes 1, 2 or 7) or the 137-1036 strain (i.e., the 137-1036 barcode) or into an E. coli strain which was subsequently used to transfer the cassettes into the 137 strains via conjugation. The presence of the homology arms on each end of the cassettes served to facilitate insertion of the cassettes into the desired sites in the genome of the tested strains according to the protocol described in Hmelo et al., “Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange,” Nat. Protoc. November 2015, which is herein incorporated by reference.

The resulting non-transgenic, uniquely barcoded strains were used in several plant experiments in order to test the utility of the described relative abundance assay.

In a first set of experiments, a set of twelve plants were each individually inoculated with a parental 137 strain (barcode 1) and a separate set of 12 plants were each individually inoculated with the 137-1036 see FIG. 7). The 137 and 137-1036 strains in this set of experiments did not contain native barcode polynucleotide cassettes and were used in a colonization assay as described in section 3 of Example 1. In summary, colonization of roots of the plants by the inoculated microbe(s) (i.e., 137 and 136-1037) were assessed using a qPCR method as described below.

One day after planting of seeds, 1 ml of microbial overnight culture (SOB media) was drenched right at the spot of where the seed was located. 1 mL of this overnight culture was roughly equivalent to about 10⁹ cfu, varying within 3-fold of each other, depending on which strain was being used. Each seedling was fertilized 3× weekly with 50 mL modified Hoagland's solution supplemented with either 2.5 mM or 0.25 mM ammonium nitrate. At four weeks after planting, root samples were collected for DNA extraction. Soil debris were washed away using pressurized water spray. These tissue samples were then homogenized using QIAGEN Tissuelyzer and the DNA was then extracted using QIAmp DNA Mini Kit (QIAGEN) according to the recommended protocol. qPCR assay was performed using Stratagene Mx3005P RT-PCR on these DNA extracts using primers that were designed (using NCBI's Primer BLAST) to be specific to a locus in each microbe's genome.

A second and third set of experiments were also conducted in order to test the efficacy of the Coco-seq assay for determining relative abundance and how it compared to the colonization assay performed in the first set of experiments.

In the second set of experiments, a set of twelve plants were each individually inoculated with pooled inoculum containing a parental 137 strain containing the barcode 1 cassette, the parental 137 strain containing the barcode 2 cassette and the parental 137 strain containing the barcode 7 cassette in a 100.10:1 ratio (see FIG. 8).

In the third set of experiments, a set of twelve plants were each individually inoculated with pooled inoculum containing the parental 137 strain containing the barcode 1 cassette, the parental 137 strain containing the barcode 2 cassette, the parental 137 strain containing the barcode 7 cassette and the 137-1036 strain containing the barcode 4 cassette in a 1:1:1:1 ratio (see FIG. 9).

For the second and third sets of experiments described above, a colonization assay similar to the one described in Example 17 of WO2019084342, which is herein incorporated by reference, was subsequently conducted. Briefly, plants in each set of experiments were inoculated with about 10⁹ cells of a strain to be tested after germination and allowed to grow for 21 days, after which they were harvested. Samples of combined seminal root and first node were taken from about 1-2 inches below the crown of the plant. After collection, samples were lysed, DNA was isolated and subjected to PCR (0.2 μl KARA 3G polymerase, 12.5 μl 2×KAPA Plant PCR Buffer, 7.8 μl molecular-grade water, 1.5 μl DNA template, 1.5 μl of 338R and 806R primers with Illumina NGS adapter sequences (10 μM)). The PCR protocol used is shown in Table D. The amplicons generated from the PCR reactions were then prepared and sequenced according to the sequencing platform recommendations (i.e., Illumina MiSeq®) Sequencing reads were then analyzed as described below.

TABLE D Coco-Seq PCR program. Step No. of Cycles Temp (Celsius) Time Initial Denaturation 1 95 3 minutes Melt 25 95 20 seconds Anneal 55 15 seconds Extension 72 30 seconds

The percent relative abundance of the specific barcode containing strains from each plant in each experiment was determined by a custom analysis pipeline. This pipeline consisted of processing raw NGS reads produced on the Illumina MiSeq platform (2×75 bp) for quality and length, identifying barcode sequences within the read, and matching observed barcode sequences to known barcode sequences via the Smith-Waterman algorithm. It should be noted that the percent relative abundance of the specific strains on day 0 was also determined for each strain in each experiment by isolating DNA from the inoculum.

As shown in FIG. 7, using the qPCR of unbarcoded strains (i.e., 137 and 137-1036 from the first set of experiments), the number of copies of 137-1036 were, on average, less than the copies of wild-type 137 strain. The whiskers on the box plots in FIG. 7 nearly completely overlap, and the second highest single plant measurement comes from the mutant (top dot on 137-1036 side of graph). While this assay can suggest that the mutant colonizes less abundantly than wildtype, the variability is still high.

FIG. 8 shows that the 137 strain present at the highest concentration in the pooled mixture (i.e., the 137 strain labelled with barcode 1) used to inoculate the 12 twelve plants used in the experiment had a higher percentage of sequence reads than did the 137 strain present at the second highest concentration in the inoculum (i.e., the 137 strain labelled with barcode 2). The 137 strain present at the lowest concentration in the inoculum (i.e., the 137 strain labelled with barcode 7) was shown to have the lowest percentage of sequence reads relative to the percent sequence reads of the other two 137 strains tested. Accordingly, FIG. 8 shows that the colonization assay of the present example was highly sensitive and could detect relative differences (over 2 logs) in colonization capacity of strains used to simultaneously inoculate plant roots using the native barcode polynucleotide cassettes described throughout this disclosure.

Like FIG. 8, FIG. 9 compares the colonization of wildtype 137; however, FIG. 9 compares colonization by a pooled, barcoded mixture of strains, included 3 different barcoded variants of wildtype 137 and 1 barcoded variant of 137-1036. The Coco-seq assays of the present example using native barcode polynucleotide cassettes were highly sensitive and could easily detect the poor colonization phenotype of the 137-1036 strain as compared to any of the parental 137 strains when the 4 strains were mixed and used to inoculate and grow on the same set of plants. In FIG. 7, absolute colonization counts are determined and compared across different plant samples. This leads to the appearance that 137-1036 may occasionally colonize better than wildtype (see whisker and outliers at top of 137-1036 column of FIG. 7). However, in every single plant in FIG. 9, 137-1036 unambiguously colonizes less effectively than wildtype. Accordingly, in the final analysis, the colonization assays presented in this example can be used to rank strains by determining each strain's abundance relative to other new isolates as well as against ladder strains of known colonization ability. Further, the results also showed that the colonization assay described in this example can produce consistent results between plant samples and thereby enables a more accurate comparison of colonization phenotypes with fewer treatments (and fewer plants per treatment).

Conclusions

In the present colonization assays, as opposed to the workflow of Example 1, all of the strains are first domesticated, and then the rest of the data is generated utilizing a relative abundance approach.

It should be noted that the current methodology can be utilized on mutant strains and is not applicable to merely wild type colonization assays.

As can be seen in FIG. 2B, there are significant numbers of plants required for the process of determining absolute abundance as described in Example 1. In some scenarios, the colonization assays based upon absolute colonization numbers can only statistically discriminate colonization differences that exceed SOX. Unlike with absolute colonization, where 240-800 plants are required to investigate 19 strains (FIG. 2B), one of the goals of the relative colonization assays described in this example is to reduce the number of plants needed to investigate a plurality of different strains such that, for example, dozens of strains could, theoretically, be investigated in as few as 1 plant. That's >100× fewer plants and >100× fewer microbial genomic DNA preparations. Furthermore, if only relative measurements are desired, genomic preparations could be simplified. While absolute colonization quantification depends on getting every shred of bacterial DNA from a sample, the relative colonization quantification described in this example depended upon obtaining representative samples of the tested microbes. As long as a representative genomic DNA sample was acquired, it was irrelevant how much of the population was harvested.

In view of the data provided in this example, the described colonization assays utilizing natural barcodes and based upon relative colonization numbers can be used to pare down the colonization workflows of Example 1 and lead to higher resolution.

It should be noted that in future experiments, the desired or target loci of native barcode polynucleotide cassettes will be chosen to meet the constraints discussed below in order to avoid bias often introduced in next-generation sequencing (NGS) in order to potentially further improve the Coco-seq assay.

A common issue arising from next-generation sequencing is bias introduced in sequencing coverage due to distance from the origin of replication. Genomic regions near the origin of replication can have a higher copy number in dividing cells and thus appear more frequently in the sequencing results as described in Wetmore K M, et al., “Rapid Quantification of Mutant Fitness in Diverse Bacteria by Sequencing Randomly Bar-Coded Transposons,” mBio May 2015, 6 (3) e00306-15, which is herein incorporated by reference. FIG. 5 provides an illustration of this issue.

In order to avoid this issue, the native barcode polynucleotide cassettes in the method in general will be designed to be inserted into tested strains such that a semi-constant distance from the origin of replication of the bacterial chromosome in each strain will be maintained. In other words, in each strain, a polynucleotide cassette will be inserted into a locus in the genome of said strain that is the same or nearly the same distance from an origin of replication of a genetic element (e.g., chromosome) as the distance from the origin of replication for insertion of each other polynucleotide cassette in each other strain. This can be done to minimize the amplification bias that can stem from location of a native barcode as provided herein in the genome of a strain.

In addition to considering distance to the origin of replication, the insertion regions for each cassette will be further identified by finding a non-coding region between two coding DNA segments which are transcribed in alternate directions and separated by a terminator region (see FIG. 6A). The goal in targeting native barcode cassette insertions to such locations is to minimize the possibility of unanticipated alterations of cell behavior. Bacterial genomes contain intragenic regions that may initially appear to be silent regions where a barcode insertion could have no effect. One such possible region includes the 321-base pair interval between rplL and rpoB in Escherichia coli. While this region might initially appear to be silent, it has been shown to be an untranslated regulatory in the middle of a multi-gene operon (www.ncbi.nlm.nih.gov/pubmed/?term=2437101). If a barcode were to be inserted here, there is a reasonable chance of inadvertently interfering with the levels of expression of the critical rpoB gene. By focusing in the regions beside transcriptional terminators, the chance of interfering with gene regulation will be minimized. Cognizant of the fact that desirable insertions are in actively transcribed regions between a genes's stop codon and its terminator sequence, avoidance of introducing any new start codons will be careful controlled in order to avoid translation of any native barcode cassettes. Consequently, native barcode cassettes will be targeted, complete with the 338R/806R universal primer binding sites (e.g., the native barcode polynucleotide cassettes described in this example) to one side of a terminator (see FIG. 6B) such that the transcription of the barcode will only occur in one direction as opposed to two. In addition, stop codons at the end of either gene will cease translation of the RNA sequences before a ribosome may encounter the specific native barcode sequence.

Example 3: Plant Colonization Assays Utilizing Natural Microbial Barcodes to Determine Absolute Abundance Objective

The following example is a proof of concept of plant colonization assays that can utilize natural microbial barcodes in order to determine absolute abundance of microbes in plant samples.

Methods and Results

In order to determine the absolute abundance of microbes on a plant using natural or native microbial barcodes as provided herein, a natural barcode polynucleotide cassette comprising the universal primer binding sites of 338R as the forward primer binding site and 806R as the reverse primer binding site with a barcode sequence located therebetween that was unique for the 137 strain was generated as described in Example 2. Additionally, a control barcode was generated that comprised the same universal primer binding sites as the 137 strain specific natural barcode polynucleotide but contained a distinct barcode sequence located between the forward and reverse universal primer binding sites. The distinct barcode sequence was selected using the parameters described in Example 2 and was based on sequences naturally occurring in the sea otter genome.

As a proof of concept, once the 137-specific barcode and the sea otter control barcode were generated, each was added to a preparation of plant gDNA and subjected to the amplification and sequencing protocols and analysis described in Example 2. The 137-specific barcode DNA was added at a known level, 10⁵ copies, while the sea otter control barcode DNA was spiked in at a number of different amounts (i.e., 10⁶, 10³, and 10⁰ copies, see closed light squares in FIG. 10).

As can be seen from the trendline and the closed and open squares in FIG. 10, the 137-specific barcode DNA was predicted to be at a level of 1.26×10⁵ copies (light open square), only a little off from the actual value (i.e., 10⁵ copies) added to the plant gDNA preparation. This result was further corroborated by performing the assay again using a second ladder with different amounts (i.e., 10¹, 10⁴ and 10⁷ copies, see dark closed circles) of the sea otter control barcode DNA, where the 137 barcode was predicted to be 1.34×10⁵ copies (open dark circle FIG. 10).

Conclusions

The results shown in FIG. 10 indicate that the natural barcode polynucleotide cassettes described throughout this disclosure can be used in colonization assays to determine absolute abundance of one or more microbial strains growing on a plant by spiking into gDNA preparations from the plant inoculated with the one or more microbial strains, a ladder of known amounts of control barcode DNA that comprises primer binding sites shared by natural barcodes present in the one or more strains and determining where the read counts for the one or more microbial strains fall on a standard curve generated from the control barcode DNA. It should be noted that each strain, variant or species inoculated onto a plant can contain natural barcodes with universal primer binding sites unique to the barcode for that strain, species or variant and standard curves can be generated for determining absolute abundance by spiking in known amounts of control barcode DNA that share the universal primer binding sites unique to the barcode for a specific strain, species or variant.

Example 4: Plant Colonization Assays Utilizing Natural Microbial Barcodes to Determine Absolute Abundance of Each Microbe in a Mixed Population of Strains Objective

The following example is a proof of concept of plant colonization assays that can utilize natural microbial barcodes in order to determine absolute abundance or total abundance of each microbe present in a mixed population of microbes in plant samples where microbes within the population contain different native barcode sequences. In contrast to Example 3 where the absolute abundance was determined by spiking barcode sequences into gDNA preparations, this example was performed to show the utility of calculating absolute abundance using similar concepts as discussed in Example 3 but performed in strains containing natural barcode sequences.

Methods and Results

In order to determine the absolute abundance of each strain of microbe in a mixed population of microbes on a plant using natural or native microbial barcodes as provided herein, a natural or native barcode polynucleotide cassette comprising the universal primer binding sites of 338R as the forward primer binding site and 806R as the reverse primer binding site with a native or natural barcode sequence located therebetween that was unique to each strain in the mixed population was generated as described in Example 2. More specifically, a natural or native barcode polynucleotide cassette was designed for each of 5 strains (i.e., 137, 137-1036, 137-3933, 137-3944 and 137-2285) such that each of the natural or native barcode polynucleotide cassettes shared the same forward and reverse universal primer binding sites (i.e., 338R and 806R) separated by a native or natural barcode sequence unique to each strain. The strain specific native barcode sequences were as follows.

137 Barcode_7: (SEQ ID NO: 427) TATCCGTAAGCGAGCAAAGCGTGAGTGAAGTCTACTACGCTCTCTCAG 137-1036 Barcode_3: (SEQ ID NO: 428) TATCCGTAAGCGAGCAAAGCGTGAAACGGCACGTCCGAATCGTATCAA 137-3933 Barcode_15: (SEQ ID NO: 435) TATCCGTAAGCGAGCAAAGCGTGACAAAGATCTTCTCCTGACTGGTCT 137-3944 Barcode_17: (SEQ ID NO: 436) TATCCGTAAGCGAGCAAAGCGTGAGACCTTCAGCCTTTATCTTAACGG 137-2286 Barcode_10: (SEQ ID NO: 437) TATCCGTAAGCGAGCAAAGCGTGAGACTATCCGATTCTGCTCCTTGAT

Additionally, six (6) control barcodes were generated such that each of the 6 control barcodes comprised the same forward and reverse universal primer binding sites (i.e., 338R and 806R) as the strain specific natural or native barcode polynucleotides but each of the 6 control barcodes contained a distinct barcode sequence located between the forward and reverse universal primer binding sites. The distinct barcode sequences were selected using the parameters described in Example 2 and were based on sequences naturally occurring in the sea otter genome. The 6 Otter block control barcode sequences were as follows.

Otter Block 1: (SEQ ID NO: 438) CAAGATGCAGTGGCCGAAAACCAGGAAGCTCACGGGATGGAAGTAGCT  Otter Block 2: (SEQ ID NO: 439) CAAGATGCAGTGGCCGAAAACCAGGGCGAGGAAACAGTCTTTAATCCT Otter Block 3: (SEQ ID NO: 440) CAAGATGCAGTGGCCGAAAACCAGAGAGACCTAGCATCGTCCTCCACT Otter Block 4: (SEQ ID NO: 441) AATGAATAGGTATTTCTGGCTCGGTCATCATTTTCCTTCTCTGGGGCT Otter Block 5: (SEQ ID NO: 442) AGATTTATCCATATCATTGCATGAGCCTGTCGGTCTTATTTGGGTGGT Otter Block 6: (SEQ ID NO: 443) AAGTCCCAGCTCCCTACTTTGTGGGGACTAGAACCAGTTTGGCTCTTT

Once designed, each of the native barcode polynucleotide cassettes and Otter block control barcodes provided above were synthesized by IDT. It should be noted that during synthesis of the native barcode polynucleotide cassettes, homology arms that comprised sequence complementary to a desired locus or site in the genome of the tested strains were introduced.

The native barcode polynucleotide cassettes were each subsequently cloned into a vector (i.e., suicide vector) and transformed into an E. coli strain which was subsequently used to transfer the cassettes into the respective 137 strains via conjugation. The presence of the homology arms on each end of the cassettes served to facilitate insertion of the cassettes into the desired sites in the genome of the tested strains according to the protocol described in Hmelo et al., “Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange,” Nat Protoc. November 2015, which is herein incorporated by reference.

The resulting non-transgenic, uniquely barcoded strains were then each separately inoculated into SOB media in 5 ml tubes and the resulting cultures were grown for 24 hours at 30° C. while shaking at 200 rpm. The strains were subsequently mixed in equal ratios and diluted to a final optical density (590 nm) of 1.0. The pooled strain inoculum was then used in a colonization assay similar to the one described in Example 17 of WO2019084342, which is herein incorporated by reference.

Briefly, 10 plants (plant samples found in Table F below) were each inoculated with 1 milliliter of the pooled strain inoculum (approximately 10{circumflex over ( )}9 cells of the of the pooled barcoded strains) and allowed to grow for 21 days in a grow room, after which they were harvested. Samples of combined seminal root and first node were taken from about 1-2 inches below the crown of each of the 10 plants. After collection, samples were lysed, DNA was isolated and subjected to PCR (0.2 μl KAPA 3G polymerase, 12.5 μl 2×KAPA Plant PCR Buffer, 7.8 μl molecular-grade water, 6 μl DNA template, 1.5 μl of 338R and 806R primers with Illumina NGS adapter sequences (10 μM)) using the PCR parameters found in Table D above. The defined set of sea otter control barcode DNA (i.e., Otter blocks 1-6 in Table E) was spiked in at a different amounts (see Table E) alongside the template gDNA in the PCR reaction for each plant. The resulting PCR amplification products were then subjected to sequencing protocols and analysis described in Example 2. Briefly, the raw NGS reads produced on an Illumina MiSeq platform (2×75 bp) were processed for quality and length, identifying barcode sequences within the read, and matching observed barcode sequences to known barcode sequences via the Smith-Waterman algorithm.

TABLE E Number of copies spiked into PCR templates for each sea otter control barcode DNA. Otter Block Copies spiked into PCR template Otter_block_1 151 Otter_block_2 50 Otter_block_3 457 Otter_block_4 1370 Otter_block_5 1370 Otter_block_6 1370

Similarly to FIG. 10, the known sea otter control barcode DNA was used to generate a trendline and predict the unknown barcoded strain present in the template gDNA. These predictions were used to calculate the total abundance of individual barcoded strains in units of cells per fresh gram weight (gfw) of root tissue based on the amount of root tissue sampled and gDNA template used in the PCR reaction.

Results

Table F shows the sequencing results for each of the plants tested in the Coco-seq assays performed as described in this example. Tables G and H show the raw counts obtained for each of the native barcodes (Table G) tested as well as the otter block control barcode DNA (Table H) from the sequencing data, while Tables I and J show the number of copies of each of the native barcodes (Table I) and otter blocks (Table J) based on the number of otter block raw counts (Table H) detected. Finally, Table K shows the number of cells per gfw of each of the strains present in the pooled strain used to inoculate and grow on each of the ten plants. The cell per gfw calculations were generated in the following manner: read counts of the six otterblocks (Table H) were used with the known copy number of each otter block present in the PCR reaction to generate a least-squares linear regression model which created a function of read counts (Table F) to number of copies present in the PCR reaction (Table I). The R squared value included in Table H was a measure of the linear regression model's accuracy in making predictions. The number of copies for the PCR reaction is used along with the Tissue Factor (the amount of tissue sampled relative to the total root tissue present in the plant) of 32, and the Elution volume (the amount of liquid used to elute during the gDNA preparations of the sampled root tissue) of 100 ul to calculate the total abundance of the strain in the entire root (Table K). This was done by multiplying the number of copies in the reaction by the Tissue Factor and the Elution volume and dividing by the amount of template used as seen in the equation below:

${{cell}\mspace{14mu}{per}\mspace{14mu}{gfw}} = {{copies}\mspace{14mu}{in}\mspace{14mu}{{reaction}\left( \frac{{Tissue}\mspace{14mu}{Factor}*{Elution}\mspace{14mu}{volume}}{{Volume}\mspace{14mu}{of}\mspace{14mu}{template}\mspace{14mu}{used}} \right)}}$

Table K also showed the total number of cells per gfw for all the 137-based strain as determined using the Coco-seq assay on each plant as compared to the total number of cells per gfw for all the 137-based strains as determined using the qPCR assay using a 137 strain specific probe as described in Example 1. As can be seen in Table K, the colonization capacity of each strain varied with each of the strains showed weaker colonization capacity than parental strain 137. FIG. 11 reflects the weaker colonization capacity of select tested strains vs. parental strain 137.

TABLE F Raw sequencing results for each of the plants tested in the Coco-seq assays performed as described in this example. well plant_sample sequencing_run_sample: reads checked: number of valid reads: percent valid: x axis E01 MTC023.0007.C.001 18325 213522 212496 99.52 1 E02 MTC023.0012.C.001 18326 246223 244769 99.41 2 E03 MTC023.0021.C.001 18327 245939 244321 99.34 3 E04 MTC023.0034.C.001 18398 163557 162618 99.43 4 E05 MTC023.0044.C.001 18399 222790 221275 99.32 5 E06 MTC023.0058.C.001 18400 161116 160348 99.52 6 E07 MTC023.0066.C.001 18401 188429 187512 99.51 7 E08 MTC023.0075.C.001 18402 181101 180380 99.6 8 E09 MTC023.0082.C.001 18403 147427 146505 99.37 9 E10 MTC023.0093.C.001 18404 151858 151173 99.55 10

TABLE G Raw counts obtained for each of the native barcodes tested from the sequencing data. 137- 137- 137- 137- 137- 137- well 1036 137 2285 3933 3944 4073 4074 E01 27779 94607 440 29055 7376 5935 4775 E02 38496 107073 2098 34811 13033 7883 13351 E03 34130 91257 803 47386 14309 11092 10565 E04 20999 86765 616 18976 5860 4184 4959 E05 20276 113617 1000 50717 5695 6116 5475 E06 14293 77260 628 34929 4316 4244 3436 E07 26247 81024 730 25785 8553 5847 7337 E08 19532 76945 507 26807 4982 4283 6132 E09 11769 52830 324 10793 4647 2831 2475 E10 15440 51020 697 18419 6062 4957 5126

TABLE H Raw counts obtained for the otter block control barcode DNA from the sequencing data. otter_ladder web otter_block_1 otter_block_2 otter_block_3 otter_block_4 otter_block_5 otter_block_6 R{circumflex over ( )}2 value E01 437 15 2979 12297 16434 10307 0.914139792 E02 334 41 1375 8678 10342 7218 0.94221294 E03 355 49 2457 10518 12406 8993 0.957732777 E04 188 19 1261 5484 7838 5463 0.92613438 E05 119 40 1406 5031 6772 4983 0.947905349 E06 169 57 1007 5968 8720 5307 0.888447645 E07 314 34 2042 9371 11724 8444 0.95112447 E08 364 76 2109 11936 15830 10877 0.931235692 E09 633 0 4420 17949 21999 15787 0.954200594 E10 322 38 3362 14498 18119 13107 0.953331447

TABLE I Number of copies of each of the native barcodes based on the number of raw otter block counts detected. 137- 137- 137- 137- 137- 137- well 1036 137 2285 3933 3944 4073 4074 E01 2842.32 9680.09 45.02 2972.9 754.704755 607.2631129 488.5731027 E02 5964.54 16589.82 325.06 5393.6 2019.32445 1221.386836 2068.595161 E03 4358.74 11654.42 102.55 6051.7 1827.400647 1416.557969 1349.254863 E04 4497.29 18582.19 131.93 4064 1255.017702 896.0740725 1062.053376 E05 4894.78 27428.02 241.41 12243 1374.81714 1476.449803 1321.707435 E06 2828.71 15290.45 124.29 6912.8 854.1752595 839.9258112 680.0153363 E07 3615.99 11162.49 100.57 3552.3 1178.32721 805.5277912 1010.801677 E08 2042.62 8046.76 53.021 2803.4 521.0079772 447.9079017 641.2727652 E09 858.77 3854.96 23.642 787.56 339.0877903 206.5757552 180.5987263 E10 1373.96 4540.12 62.024 1639.1 539.4393496 441.1086862 456.1474935

TABLE J Number of otter blocks based on the number of otter block counts detected. web otter_block_1 otter_block_2 otter_block_3 otter_block_4 otter_block_5 otter_block_6 E01 44.7133918 1.534784616 304.8082247 1258.21643 1681.51002 1054.601669 E02 51.74974038 6.35251304 213.0415959 1344.56361 1602.38268 1118.352174 E03 45.33700677 6.257784032 313.7831708 1343.2525 1584.36875 1148.494935 E04 40.26 4.07 270.06 1174.49 1678.64 1169.99 E05 28.73 9.66 339.42 1214.52 1634.81 1202.93 E06 33.45 11.28 199.29 1181.12 1725.77 1050.30 E07 43.26 4.68 281.32 1291.02 1615.19 1163.31 E08 38.07 7.95 220.56 1248.24 1655.47 1137.50 E09 46.19 0.00 322.52 1309.72 1605.25 1151.96 E10 28.65 3.38 299.17 1290.13 1612.36 1166.35

TABLE K Number of cells per gfw calculated for each of the strains on each plant tested. coco_total_(—) cell_(—) 137_ probe_(—) per_gfw qPCRcell_(—) for 137 well per_gfw strains 137-1036 137 137-2285 137-3933 137-3944 137-4073 137-4074 E01 1.35E+07 9.28E+06 1.52E+06 5.16E+06 2.40E+04 1.59E+06 4.03E+05 3.24E+05 2.61E+05 E02 3.41E+07 1.79E+07 3.18E+06 8.85E+06 1.73E+05 2.S8E+06 1.08E+06 6.51E+05 1.10E+06 E03 2.09E+07 1.43E+07 2.32E+06 6.22E+06 5.47E+04 3.23E+06 9.75E+05 7.55E+05 7.20E+05 E04 2.44E+07 1.63E+07 2.40E+06 9.91E+06 7.04E+04 2.17E+06 6.69E+05 4.78E+05 5.66E+05 E05 4.07E+07 2.61E+07 2.61E+06 1.46E+07 1.29E+05 6.53E+06 7.33E+05 7.87E+05 7.05E+05 E06 2.49E+07 1.47E+07 1.51E+06 8.15E+06 6.63E+04 3.69E+06 4.56E+05 4.48E+05 3.63E+05 E07 1.93E+07 1.14E+07 1.93E+06 5.95E+06 5.36E+04 1.89E+06 6.28E+05 4.30E+05 5.39E+05 E08 1.17E+07 7.76E+06 1.09E+06 4.29E+06 2.83E+C4 1.50E+06 2.78E+05 2.39E+05 3.42E+05 E09 4.37E+06 3.33E+06 4.58E+05 2.06E+06 1.26E+04 4.20E+05 1.81E+05 1.10E+05 9.63E+04 E10 6.72E+06 4.83E+06 7.33E+05 2.42E+06 3.31E+04 8.74E+05 2.S8E+05 2.35E+05 2.43E+05

Conclusions

The results shown in Table K and FIG. 11 indicate that the natural barcode polynucleotide cassettes described throughout this disclosure can be used in colonization assays to determine total abundance of one or more microbial strains growing on a plant by spiking into gDNA preparations from plants inoculated with the one or more microbial strains, known amounts of control barcode DNA that comprises primer binding sites shared by natural barcodes present in the one or more strains and determining where the copy number for the one or more microbial strains can be calculated via a least-squares linear regression model generated from the control barcode DNA. It should be noted that each strain, variant or species inoculated onto a plant can contain natural barcodes with universal primer binding sites unique to the barcode for that strain, species or variant and trendlines can be generated for determining total abundance by spiking in known amounts of control barcode DNA that share the universal primer binding sites unique to the barcode for a specific strain, species or variant.

TABLE 28 Further Descriptions of Strains from the Disclosure Strain Accession ID Lineage Mutagenic DNA Description Genotype Number CI006 CI006 Wildtype parent Kosakonia WT 201701001 saccari CI137 CI137 Wildtype parent K. variicola WT 201708001 CI910 CI910 Wildtype parent WT PTA-126585 Metakosakoma intestini CI8 CI8 Wildtype parent WT PTA-126582 Paraburkholderia tropica CI41 CI41 Wildtype parent WT PTA-126581 Paenibacillus polymyxa CI3069 CI3069 Wildtype parent WT PTA-126583 Herbaspirillum aquaticum 6-403 Mutant Disruption of nifL gene with ΔnifL::Prm1, ΔglnE- 201708004 of a fragment of the region AR_KO2 CI006 upstream of the lpp gene inserted (Prm1) upstream of nifA. Deletion of the 1647 bp after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate-ammonia-ligase adenylyltransferase (ΔglnE- AR_KO2). 6-2425 Mutant Disruption of nifL gene with ΔnifL::Prm1, PTA-126575 of a fragment of the region AglnD_UT_truncation CI006 upstream of the lpp gene inserted (Prm1) upstream of nifA. Deletion of the 987 bp after the start codon of the glnD gene containing the uridylyltransferase (UT) domain of the bifunctional uridylyltransferase/uridylyl- removing enzyme (ΔglnD- UT_truncation) 6-2634 Mutant Disruption of nifL gene with ΔnifL::Prm1, PTA-126576 of a fragment of the region ΔglnD_UT_deactivation CI006 upstream of the lpp gene inserted (Prm1) upstream of nifA. Deactivation of the uridylyltransferase (UT) domain of the bifunctional uridylyltransferase/uridylyl- removing enzyme, glnD, by mutating amino acid residues 90 and 91 from GG to DV as well as residue 104 from D to A. 137-1034 Mutant Deletion of the 1647 bp after ΔglnE-AR_KO2 201712001 of the start codon of the glnE CI137 gene containing the adenylyl-removing domain of glutamate-ammonia-ligase adenylyltransferase (ΔglnE- AR_KO2). 137-2084 Mutant Disruption of nifL gene with ΔnifL::Prm1.2 ΔglnE- of a fragment of the region AR_KO2 CI137 upstream of the cspE gene inserted (Prm1.2) upstream of nifA. Deletion of the 1647 bp after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate- ammonia-ligase adenylyltransferase (ΔglnE- AR_KO2). 137-1968 Deletion of the native dctA1 ΔnifL::P8.2, ΔglnE- PTA-126577 promoter and insertion of a AR_KO2 fragment (Prm8.2), found 77 bp after the start codon of nlpI to 376 bp after the start codon of nlpI, directly upstream of dctA1. Deletion of the 1647 bp after the start codon of the glnE gene containing the adenylyl- removing domain of glutamate-ammonia-ligase adenylyltransferase (ΔglnE- AR_KO2). 137-2219 Mutant Disruption of nifL gene with ΔnifL::Prm1.2 ΔglnE- PTA-126578 of a fragment of the region AR_KO2, CI137 upstream of the cspE gene ΔglnD_ACT12_truncation inserted (Prm1.2) upstream of nifA. Deletion of the 1647 bp after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate- ammonia-ligase adenylyltransferase (ΔglnE- AR_KO2). Deletion of the 546 bp before the stop codon of the glnD gene containing the ACT1/2 domain of the bifunctional uridylyltransferase/uridylyl- removing enzyme (ΔglnD- ACT12_truncation) 137-2237 Mutant Disruption of nifL gene with ΔnifL::Prm1.2, ΔglnE- PTA-126579 of a fragment of the region AR_KO2, glsA2::Prm1.2 CI137 upstream of the cspE gene inserted (Prm1.2) upstream of nifA. Deletion of the 1647 bp after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate- ammonia-ligase adenylyltransferase (ΔglnE- AR_KO2). Deletion of the native glsA2 promoter and insertion of a fragment (Prm1.2) directly upstream of the glsA2 CDS. 137-2285 Mutant Disruption of nifL gene with ΔnifL::Prm1.2, ΔglnE- PTA-126580 of a fragment of the region AR_KO2, rpoN::Prm1.2 CI137 upstream of the cspE gene inserted (Prm1.2) upstream of nifA. Deletion of the 1647 bp after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate- ammonia-ligase adenylyltransferase (ΔglnE- AR_KO2). Deletion of the native rpoN promoter and insertion of a fragment (Prm1.2) directly upstream of the rpoN CDS. 910-3994 Mutant Disruption of nifL gene with ΔnifL::Prm2.1, ΔglnE- PTA-126588 of a fragment of the region AR_KO2, glsA2::Prm1.1 CI910 upstream of the rmF gene inserted (Prm2.1) upstream of nifA. Deletion of the 1647 bp after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate- ammonia-ligase adenylyltransferase (ΔglnE- AR_KO2). Deletion of the native glsA2 promoter and insertion of a fragment upstream of the csrA gene (Prm1.1) directly upstream of the glsA2 CDS. 910-3963 Mutant Disruption of nifL gene with ΔnifL::Prm2.1, ΔglnE- PTA-126586 of a fragment of the region AR_KO2, CI910 upstream of the rmF gene ΔglnD_UT_truncation inserted (Prm2.1) upstream of nifA. Deletion of the 1647 bp after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate- ammonia-ligase adenylyltransferase (ΔglnE- AR_KO2). Deletion of the 987 bp after the start codon of the glnD gene containing the uridylyltransferase (UT) domain of the bifunctional uridylyltransferase/uridylyl- removing enzyme (ΔglnD- UT_truncation) 910-3655 Mutant Disruption of nifL gene with ΔnifL::Prm2.1, ΔglnE- PTA-126584 of a fragment of the region AR_KO2 CI910 upstream of the rmF gene inserted (Prm2.1) upstream of nifA. Deletion of the 1647 bp after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate- ammonia-ligase adenylyltransferase (AglnE- AR_KO2), 910-3961 Mutant Disruption of nifL gene with ΔnifL::Prm2.1, PTA-126587 of a fragment of the region ΔglnD_UT_truncation CI910 upstream of the rmF gene inserted (Prm2.1) upstream of nifA. Deletion of the 987 bp after the start codon of the glnD gene containing the uridylyltransferase (UT) domain of the bifunctional uridylyltransferase/uridylyl- removing enzyme (ΔglnD- UT_truncation).

Numbered Embodiments of the Disclosure

Other subject matter contemplated by the present disclosure is set out in the following numbered embodiments:

1. A method of barcoding a host cell, the method comprising:

-   -   (a) obtaining a donor cell;     -   (b) selecting and isolating a first nucleotide sequence in the         genome of the donor cell, selecting and isolating a second         nucleotide sequence in the genome of the donor cell, and         selecting and isolating a third nucleotide sequence in the         genome of the donor cell;     -   (c) creating a native barcode polynucleotide cassette comprising         the first, second, and third nucleotide sequences oriented 5′ to         3′ as:         -   (i) the first nucleotide sequence, wherein the first             nucleotide sequence is a forward primer binding site,         -   (ii) the second nucleotide sequence, wherein the second             nucleotide sequence is a barcode, and         -   (iii) the third nucleotide sequence, wherein the third             nucleotide sequence is a reverse primer binding site; and     -   (d) inserting the native barcode polynucleotide cassette into         the genome of a host cell of the same species as the donor cell         in (a); wherein the nucleotide sequences of (i), (ii), and (iii)         are native to the host cell.         2. The method of embodiment 1, wherein the donor cell and the         host cell are selected from a bacterial cell, a fungal cell, a         plant cell, an animal cell, a protozoal cell, and an insect         cell.         3. The method of embodiment 2, wherein each of the donor cell         and the host cell is a bacterial cell.         4. The method of any one of the above embodiments, wherein the         nucleotide sequence of at least one of (i), (ii), and (iii) is         isolated from ribosomal DNA or internal transcribed spacer (ITS)         DNA.         5. The method of any one of the above embodiments, wherein the         nucleotide sequences of at least one of (i), (ii), and (iii) are         isolated from 16S rDNA or 18S rDNA.         6. The method of any one of the above embodiments, wherein at         least one of the primer binding sites is selected from 8F, 27F,         CCF, 357F, 515F, 533F, 16S.1100.F16, 804F, 1237F, 338R, 519R,         CDR, 806R, 907R, 1100R, 1391R, 1392R, 1492R0), and 1492R(s).         7. The method of any one of the above embodiments, wherein the         first nucleotide sequence in the genome of the donor cell and         the third nucleotide sequence in the genome of the donor cell         occur in the same orientation as one another.         8. The method of any one of the above embodiments, wherein at         least one of (i), (ii), and (iii) do not naturally occur         immediately adjacent to one another in the genome of the host         cell.         9. The method of any one of the above embodiments, wherein the         barcode consists of fewer than 100 nucleotides.         10. The method of any one of the above embodiments, wherein one         or more of the primer binding sites consist of fewer than 30         nucleotides.         11. The method of any one of the above embodiments, wherein the         nucleotide sequences of the primer binding site and the barcode         collectively consist of fewer than 160 nucleotides.         12. The method of any one of the above embodiments, wherein the         barcode comprises a constant barcode region and a variable         barcode region.         13. The method of any one of the above embodiments, wherein the         insertion of the cassette into the genome of the host cell         introduces one or more stop codons in either orientation in the         host cell.         14. The method of any one of the above embodiments, wherein the         cassette is inserted in the genome of the host cell between two         coding regions separated by a terminator region.         15. The method of embodiment 14, wherein the cassette is         inserted between the terminator region and one of the two coding         regions.         16. The method of any one of the above embodiments, wherein the         host cell in (d) is of the same strain as the donor cell in (a).         17. The method of embodiment 3, wherein the bacterial host cell         is transgenic.         18. The method of embodiment 3, wherein the bacterial host cell         is a non-intergeneric remodeled bacterium.         19. The method of embodiment 18, wherein the non-intergeneric         remodeled bacterial host cell is, or is derived from, a         bacterium selected from Table 1.         20. The method of embodiment 19, wherein the non-intergeneric         remodeled bacterium comprises at least one genetic variation         introduced into at least one gene, or non-coding polynucleotide,         of the nitrogen fixation or assimilation genetic regulatory         network, such that the modified bacterium is capable of fixing         atmospheric nitrogen in the presence of exogenous nitrogen.         21. The method of any one of embodiments 3 or 17-20, wherein the         bacterial host cell is capable of fixing atmospheric nitrogen in         the presence of exogenous nitrogen.         22. The method of any one of embodiments 3 or 17-21, wherein the         bacterial host cell comprises at least one genetic variation         introduced into at least one gene, or non-coding polynucleotide,         of the nitrogen fixation or assimilation genetic regulatory         network.         23. The method of any one of embodiments 3 or 17-22, wherein the         bacterial host cell comprises an introduced control sequence         operably linked to at least one gene of the nitrogen fixation or         assimilation genetic regulatory network.         24. The method of any one of embodiments 3 or 17-23, wherein the         bacterial host cell comprises a heterologous promoter operably         linked to at least one gene of the nitrogen fixation or         assimilation genetic regulatory network.         25. The method of any one of embodiments 3 or 17-24, wherein the         bacterial host cell comprises at least one genetic variation         introduced into a member selected from the group consisting of:         nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine         synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide         encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY,         nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB,         nifQ, a gene associated with biosynthesis of a nitrogenase         enzyme, and combinations thereof.         26. The method of any one of embodiments 3 or 17-25, wherein the         bacterial host cell comprises at least one genetic variation         introduced into at least one gene, or non-coding polynucleotide,         of the nitrogen fixation or assimilation genetic regulatory         network that results in one or more of: increased expression or         activity of NifA or glutaminase; decreased expression or         activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT,         AmtB; decreased adenylyl-removing activity of GlnE; and         decreased uridylyl-removing activity of GlnD.         27. The method of any one of embodiments 3 or 17-23, wherein the         bacterial host cell comprises a mutated nifL gene comprising a         heterologous promoter in said nifL gene.         28. The method of any one of embodiments 3 or 17-23, wherein the         bacterial host cell comprises a mutated glnE gene that results         in a truncated GlnE protein lacking an adenylyl-removing (AR)         domain.         29. The method of any one of embodiments 3 or 17-23, wherein the         bacterial host cell comprises a mutated amtB gene that results         in the lack of expression of said amtB gene.         30. The method of any one of embodiments 3 or 17-23, wherein the         bacterial host cell comprises at least one of: a mutated nifL         gene comprising a heterologous promoter in said nifL gene: a         mutated glnE gene that results in a truncated GlnE protein         lacking an adenylyl-removing (AR) domain; a mutated amtB gene         that results in the lack of expression of said amtB gene; and         combinations thereof.         31. The method of any one of embodiments 3 or 17-23, wherein the         bacterial host cell comprises a mutated nifL gene comprising a         heterologous promoter in said nifL gene and a mutated glnE gene         that results in a truncated GlnE protein lacking an         adenylyl-removing (AR) domain.         32. The method of any one of embodiments 3 or 17-23, wherein the         bacterial host cell comprises a mutated nifL gene comprising a         heterologous promoter in said nifL gene, a mutated glnE gene         that results in a truncated GlnE protein lacking an         adenylyl-removing (AR) domain, and a mutated amtB gene that         results in the lack of expression of said amtB gene.         33. The method of any one of embodiments 3 or 17-32, wherein the         bacterial host cell is selected from Paraburkholderia tropica,         Paraburkholderia xenovorans, Herbaspirillum seropedicae,         Herbospirullum frisingense, Pseudomonas protegens, Pseudomonas         syringae, Pseudomonas benzenivorans, Metakosakonia massiliensis,         Metakosakonia intestinii, Rahnella aquatilis, Klebsiella         variicola, Achromobacter spiritinus, Achromobacter marplatensis,         Microbacterium murale, Kluyvera intermedia, Kosakonia         pseudosacchari, Enterobacter sp., Azospirillum lipoferum, and         Kosakonia sacchari.         34. The method of any one of embodiments 3 or 17-33, wherein the         bacterial host cell is endophytic, epiphytic, or rhizospheric.         35. The method of any one of embodiments 3 or 17-34, wherein the         bacterial host cell is selected from: a bacterium deposited as         ATCC PTA-126575, a bacterium deposited as ATCC PTA-126576, a         bacterium deposited as ATCC PTA-126577, a bacterium deposited as         ATCC PTA-126578, a bacterium deposited as ATCC PTA-126579, a         bacterium deposited as ATCC PTA-126580, a bacterium deposited as         ATCC PTA-126581, a bacterium deposited as ATCC PTA-126582, a         bacterium deposited as ATCC PTA-126583, a bacterium deposited as         ATCC PTA-126584, a bacterium deposited as ATCC PTA-126585, a         bacterium deposited as ATCC PTA-126586, a bacterium deposited as         ATCC PTA-126587, a bacterium deposited as ATCC PTA-126588, a         bacterium deposited as NCMA 201701001, a bacterium deposited as         NCMA 201701002, a bacterium deposited as NCMA 201701003, a         bacterium deposited as NCMA 201708004, a bacterium deposited as         NCMA 201708003, a bacterium deposited as NCMA 201708002, a         bacterium deposited as NCMA 201708001, a bacterium deposited as         NCMA 201712001, and a bacterium deposited as NCMA 201712002.         36. The method of any one of embodiments 3 or 17-35, wherein the         bacterial host cell comprises a nucleic acid sequence that         shares at least about 95% sequence identity with a nucleic acid         sequence selected from SEQ ID NOs: 177-260 and 296-303.         37. The method of any one of embodiments 3 or 17-35, wherein the         bacterial host cell comprises a nucleic acid sequence that         shares at least about 99% sequence identity with a nucleic acid         sequence selected from SEQ ID NOs: 177-260 and 296-303.         38. The method of any one of embodiments 3 or 17-35, wherein the         bacterial host cell comprises a nucleic acid sequence selected         from SEQ ID NOs: 177-260 and 296-303.         39. A composition comprising:     -   (a) one or more modified cells comprising, in their genome.         -   (i) a naturally occurring first nucleotide sequence, second             nucleotide sequence, and third nucleotide sequence, and         -   (ii) a native barcode polynucleotide cassette comprising the             first, second, and third nucleotide sequences oriented 5′ to             3′ as:             -   (1) the first nucleotide sequence, wherein the first                 nucleotide sequence is a forward primer binding site,             -   (2) the second nucleotide sequence, wherein the second                 nucleotide sequence is a barcode, and             -   (3) the third nucleotide sequence, wherein the third                 nucleotide sequence is a reverse primer binding site;                 wherein the first, second, and third nucleotide                 sequences of (ii) are in a different proximity to one                 another as compared to the naturally occurring sequences                 of (i).                 40. The composition of embodiment 39, wherein the                 barcode is unique to different species of the one or                 more modified cells.                 41. The composition of embodiment 39, wherein the                 barcode is unique to different strains of the one or                 more modified cells.                 42. The composition of any one of embodiments 39-41,                 wherein the one or more modified cells comprise a                 homogenous population of cells.                 43. The composition of any one of embodiments 39-41,                 wherein the one or more modified cells comprise a                 heterogeneous population of cells.                 44. The composition of embodiment 43, wherein the                 heterogeneous population of cells comprises two or more                 different species.                 45. The composition of embodiment 43, wherein the                 heterogeneous population of cells comprises two or more                 different strains.                 46. The composition of embodiment 44, wherein the                 heterogeneous population of cells comprises two or more                 strains of each species.                 47. The composition of any one of embodiments 39-46,                 wherein the one or more modified cells is selected from                 a bacterial cell, a fungal cell, a plant cell, an animal                 cell, a protozoal cell, and an insect cell.                 48. The composition of any one of embodiments 39-47,                 wherein the one or more modified cells is a bacterial                 cell.                 49. The composition of any one of embodiments 39-48,                 wherein the nucleotide sequence of at least one of (1),                 (2), or (3) is isolated from ribosomal DNA or internal                 transcribed spacer (ITS) DNA.                 50. The composition of any one of embodiments 39-49,                 wherein the nucleotide sequence of at least one of (1),                 (2), or (3) is 16S rDNA or 18S rDNA.                 51. The composition of any one of embodiments 39-50,                 wherein at least one of the primer binding sites is                 selected from 8F, 27F, CCF, 357F, 515F, 533F,                 16S.1100.F16, 804F, 1237F, 338R, 519R, CDR, 806R, 907R,                 1100R, 1391R, 1392R, 1492R(l), and 1492R(s).                 52. The composition of any one of embodiments 39-51,                 wherein the naturally occurring first and third                 nucleotide sequences in the genome of the modified cells                 occur in the same orientation as one another.                 53. The composition of any one of embodiments 39-52,                 wherein at least one of the naturally occurring first,                 second, or third nucleotide sequences do not occur                 immediately adjacent to one another in the genome of the                 modified cell.                 54. The composition of any one of embodiments 39-53,                 wherein the barcode consists of fewer than 100                 nucleotides.                 55. The composition of any one of embodiments 39-54,                 wherein one or more of the primer binding sites consist                 of fewer than 30 nucleotides.                 56. The composition of any one of embodiments 39-55,                 wherein the nucleotide sequences of the primer binding                 sites and the barcode collectively consist of fewer than                 160 nucleotides.                 57. The composition of any one of embodiments 39-56,                 wherein the barcode comprises a constant barcode region                 and a variable barcode region.                 58. The composition of embodiment 57, wherein the                 constant barcode region of the barcode is the same in                 each of the one or more modified cells.                 59. The composition of embodiment 57 or 58, wherein the                 variable barcode region of the barcode is different in                 each of the one or more modified cells, with only cells                 of the same strain, species, or other categorical                 distinction sharing the same variable region.                 60. The composition of any one of embodiments 39-59,                 wherein the cassette possesses one or more stop codons                 in either orientation in the one or more modified cells.                 61. The composition of any one of embodiments 39-60,                 wherein the cassette is present in the genome of the one                 or more modified cells between two coding regions                 separated by a terminator region.                 62. The composition of embodiment 61, wherein the                 cassette is present between the terminator region and                 one of the two coding regions.                 63. The composition of any one of embodiments 39-62,                 wherein the cassette is present in the genome of each of                 the one or more modified cells at a set distance from an                 origin of replication.                 64. The composition of embodiment 63, wherein the set                 distance from the origin of replication is similar or                 identical in each of the two or more cells.                 65. The composition of embodiment 48, wherein the one or                 more modified bacteria are transgenic.                 66. The composition of embodiment 48, wherein the one or                 more modified bacteria are non-intergeneric remodeled                 bacteria.                 67. The composition of embodiment 48, wherein the one or                 more modified bacteria comprise a population of                 transgenic bacteria.                 68. The composition of embodiment 48 or 66, wherein the                 one or more modified bacteria comprise a population of                 non-intergeneric remodeled bacteria.                 69. The composition of embodiment 66, wherein the                 non-intergeneric remodeled bacteria comprise, or are                 derived from, a bacterium selected from Table 1.                 70. The composition of embodiment 69, wherein the                 non-intergeneric remodeled bacteria comprise at least                 one genetic variation introduced into at least one gene,                 or non-coding polynucleotide, of the nitrogen fixation                 or assimilation genetic regulatory network, such that                 the modified bacterium is capable of fixing atmospheric                 nitrogen in the presence of exogenous nitrogen.                 71. The composition of embodiment 48, wherein the                 bacterial cell is capable of fixing atmospheric nitrogen                 in the presence of exogenous nitrogen.                 72. The composition of embodiment 48, wherein the                 bacterial cell comprises at least one genetic variation                 introduced into at least one gene, or non-coding                 polynucleotide, of the nitrogen fixation or assimilation                 genetic regulatory network.                 73. The composition of any one of embodiments 48 or                 65-72, wherein the bacterial cell comprises an                 introduced control sequence operably linked to at least                 one gene of the nitrogen fixation or assimilation                 genetic regulatory network.                 74. The composition of any one of embodiments 48 or                 65-73, wherein the bacterial cell comprises a                 heterologous promoter operably linked to at least one                 gene of the nitrogen fixation or assimilation genetic                 regulatory network.                 75. The composition of any one of embodiments 48 or                 65-74, wherein the bacterial cell comprises at least one                 genetic variation introduced into a member selected from                 the group consisting of: nifA, nifL, ntrB, ntrC,                 polynucleotide encoding glutamine synthetase, glnA,                 glnB, glnK, drat, amtB, polynucleotide encoding                 glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY,                 nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF,                 nifB, nifQ, a gene associated with biosynthesis of a                 nitrogenase enzyme, and combinations thereof.                 76. The composition of any one of embodiments 48 or                 65-75, wherein the bacterial cell comprises at least one                 genetic variation introduced into at least one gene, or                 non-coding polynucleotide, of the nitrogen fixation or                 assimilation genetic regulatory network that results in                 one or more of: increased expression or activity of NifA                 or glutaminase; decreased expression or activity of                 NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT,                 AmtB; decreased adenylyl-removing activity of GlnE; and                 decreased uridylyl-removing activity of GlnD.                 77. The composition of any one of embodiments 48 or                 65-74, wherein the bacterial cell comprises a mutated                 nifL gene comprising a heterologous promoter in said                 nifL gene.                 78. The composition of any one of embodiments 48 or                 65-74, wherein the bacterial cell comprises a mutated                 glnE gene that results in a truncated GlnE protein                 lacking an adenylyl-removing (AR) domain.                 79. The composition of any one of embodiments 48 or                 65-74, wherein the bacterial cell comprises a mutated                 amtB gene that results in the lack of expression of said                 amtB gene.                 80. The composition of any one of embodiments 48 or                 65-74, wherein the bacterial cell comprises at least one                 of: a mutated nifL gene comprising a heterologous                 promoter in said nifL gene; a mutated glnE gene that                 results in a truncated GlnE protein lacking an                 adenylyl-removing (AR) domain; a mutated amtB gene that                 results in the lack of expression of said amtB gene; and                 combinations thereof.                 81. The composition of any one of embodiments 48 or                 65-74, wherein the bacterial cell comprises a mutated                 nifL gene comprising a heterologous promoter in said                 nifL gene and a mutated glnE gene that results in a                 truncated GlnE protein lacking an adenylyl-removing (AR)                 domain.                 82. The composition of any one of embodiments 48 or                 65-74, wherein the bacterial cell comprises a mutated                 nifL gene comprising a heterologous promoter in said                 nifL gene, a mutated glnE gene that results in a                 truncated GlnE protein lacking an adenylyl-removing (AR)                 domain, and a mutated amtB gene that results in the lack                 of expression of said amtB gene.                 83. The composition of any one of embodiments 48 or                 65-82, wherein the bacterial cell is selected from                 Rahnella aquatilis, Klebsiella variicola, Achromobacter                 spiritinus, Achromobacter marplatensis. Microbacterium                 murale, Kluyvera intermedia, Kosakonia pseudosacchari,                 Enterobacter sp., Azospirillum lipoferum, and Kosakonia                 sacchari.                 84. The composition of any one of embodiments 48 or                 65-83, wherein the bacterial cell is endophytic,                 epiphytic, or rhizospheric.                 85. The composition of any one of embodiments 48 or                 65-84, wherein the bacterial cell is selected from, a                 bacterium deposited as ATCC PTA-126575, a bacterium                 deposited as ATCC PTA-126576, a bacterium deposited as                 ATCC PTA-126577, a bacterium deposited as ATCC                 PTA-126578, a bacterium deposited as ATCC PTA-126579, a                 bacterium deposited as ATCC PTA-126580, a bacterium                 deposited as ATCC PTA-126581, a bacterium deposited as                 ATCC PTA-126582, a bacterium deposited as ATCC                 PTA-126583, a bacterium deposited as ATCC PTA-126584, a                 bacterium deposited as ATCC PTA-126585, a bacterium                 deposited as ATCC PTA-126586, a bacterium deposited as                 ATCC PTA-126587, a bacterium deposited as ATCC                 PTA-126588, a bacterium deposited as NCMA 201701001, a                 bacterium deposited as NCMA 201701002, a bacterium                 deposited as NCMA 201701003, a bacterium deposited as                 NCMA 201708004, a bacterium deposited as NCMA 201708003,                 a bacterium deposited as NCMA 201708002, a bacterium                 deposited as NCMA 201708001, a bacterium deposited as                 NCMA 201712001, and a bacterium deposited as NCMA                 201712002.                 86. The composition of any one of embodiments 48 or                 65-85, wherein the bacterial cell comprises a nucleic                 acid sequence that shares at least about 95% sequence                 identity with a nucleic acid sequence selected from SEQ                 ID NOs: 177-260 and 296-303.                 87. The composition of any one of embodiments 48 or                 65-85, wherein the bacterial cell comprises a nucleic                 acid sequence that shares at least about 99% sequence                 identity with a nucleic acid sequence selected from SEQ                 ID NOs: 177-260 and 296-303.                 88. The composition of any one of embodiments 48 or                 65-85, wherein the bacterial cell comprises a nucleic                 acid sequence selected from SEQ ID NOs: 177-260 and                 296-303.                 89. A method of screening cells in a sample, the method                 comprising:     -   (a) inserting a native barcode polynucleotide cassette into the         genomes of two or more cells, wherein the cassette comprises         nucleotide sequences native to each of the two or more cells,         oriented 5′ to 3′ as.         -   (i) a forward primer binding site,         -   (ii) a barcode unique to each cell, species, or strain, and         -   (iii) a reverse primer binding site;     -   (b) applying the two or more cells to a proliferative medium;     -   (c) collecting a sample of the proliferative medium comprising         the two or more cells, and     -   (d) determining the abundance of the two or more cells in the         sample by analyzing the abundance of the barcodes.         90. The method of embodiment 89, wherein step (c) further         comprises lysing the two or more cells.         91. The method of embodiment 89 or 90, wherein the abundance is         relative abundance.         92. The method of embodiment 89 or 90, wherein the abundance is         absolute abundance.         93. The method of any one of embodiments 89-92, wherein the         abundance is determined by PCR.         94. The method of embodiment 93, wherein the PCR is selected         from multiplex-PCR, dPCR, ddPCR, and qPCR.         95. The method of any one of embodiments 89-94, wherein the         proliferative medium is selected from a liquid medium, a solid         medium, and a semi-solid medium.         96. The method of any one of embodiments 89-94, wherein the         proliferative medium is soil.         97. The method of embodiment 96, wherein the soil comprises one         or more plants or plant parts.         98. The method of embodiment 97, wherein the one or more plants         or plant parts are selected from non-leguminous plants.         99. The method of embodiment 98, wherein the one or more         non-leguminous plants is corn.         100. The method of any one of embodiments 89-99, wherein the         period of time between steps (c) and (d) is an amount of time         sufficient for at least one doubling of the two or more cells.         101. The method of any one of embodiments 89-100, wherein the         forward primer binding site and/or the reverse primer binding         site are the same in each of the two or more cells.         102. The method of any one of embodiments 89-101, wherein the GC         content of the forward primer binding site and/or the reverse         primer binding site is at least 40%.         103. The method of any one of embodiments 89-102, wherein the         two or more cells comprise a homogenous population of cells.         104. The method of any one of embodiments 89-102, wherein the         two or more cells comprise a heterogeneous population of cells.         105. The method of embodiment 104, wherein the heterogeneous         population of cells comprises two or more different species.         106. The method of embodiment 104, wherein the heterogeneous         population of cells comprises two or more different strains.         107. The method of embodiment 105, wherein the heterogeneous         population of cells comprises two or more strains of each         species.         108. The method of any one of embodiments 89-107, wherein the         two or more cells are selected from bacterial cells, fungal         cells, plant cells, animal cells, protozoal cells, and insect         cells.         109. The method of embodiment 108, wherein the two or more cells         are bacterial cells.         110. The method of any one of embodiments 89-109, wherein the         nucleotide sequence of at least one of (i), (ii), or (iii) is         isolated from ribosomal DNA or internal transcribed spacer (ITS)         DNA.         111. The method of any one of embodiments 89-110, wherein the         nucleotide sequence of at least one of (i), (ii), or (iii) is         16S rDNA or 18S rDNA.         112. The method of any one of embodiments 89-111, wherein at         least one of the primer binding sites is selected from 8F, 27F,         CCF, 357F, 515F, 533F, 16S.1100.F16, 804F, 1237F, 338R, 519R,         CDR, 806R, 907R, 1100R, 1391R, 1392R, 1492R0), and 1492R(s).         113. The method of any one of embodiments 89-112, wherein the         barcode consists of fewer than 100 nucleotides.         114. The method of any one of embodiments 89-113, wherein one or         more of the primer binding sites consist of fewer than 30         nucleotides.         115. The method of any one of embodiments 89-114, wherein the         nucleotide sequences of the primer binding sites and the barcode         collectively consist of fewer than 160 nucleotides.         116. The method of embodiment 109, wherein the two or more         bacteria are transgenic.         117. The method of embodiment 109, wherein the two or more         bacteria are non-intergeneric remodeled bacteria.         118. The method of embodiment 109, wherein the two or more         bacteria comprise a population of transgenic bacteria.         119. The method of embodiment 109, wherein the two or more         bacteria comprise a population of non-intergeneric remodeled         bacteria.         120. The method of embodiment 117, wherein the non-intergeneric         remodeled bacteria comprise, or are derived from, a bacterium         selected from Table 1.         121. The method of any one of embodiments 109 or 116-120,         wherein the two or more bacteria are capable of fixing         atmospheric nitrogen in the presence of exogenous nitrogen.         122. The method of any one of embodiments 89-121, wherein the         barcode comprises a constant barcode region and a variable         barcode region.         123. The method of embodiment 122, wherein the constant barcode         region of the barcode inserted into each of the two or more         cells is the same in each of the two or more cells.         124. The method of embodiment 122 or 123, wherein the variable         barcode region of the barcode inserted into each of the two or         more cells is different, with only cells of the same strain,         species, or other categorical distinction sharing the same         variable region.         125. The method of any one of embodiments 89-124, wherein the         insertion of the cassette into the genome of the two or more         cells introduces one or more stop codons in either orientation         in the two or more cells.         126. The method of any one of embodiments 89-125, wherein the         cassette is present in the genome of the two or more cells         between two coding regions separated by a terminator region.         127. The method of embodiment 126, wherein the cassette is         present between the terminator region and one of the two coding         regions.         128. The method of any one of embodiments 89-127, wherein the         cassette is present in the genome of each of the two or more         cells at a set distance from an origin of replication.         129. The method of embodiment 128, wherein the set distance from         the origin of replication is similar or identical in each of the         two or more cells.         130. A method of barcoding a population of host cells, the         method comprising.     -   (a) obtaining a donor cell;     -   (b) selecting and isolating a first nucleotide sequence in the         genome of the donor cell, selecting and isolating a second         nucleotide sequence in the genome of the donor cell, and         selecting and isolating a third nucleotide sequence in the         genome of the donor cell;     -   (c) creating a native barcode polynucleotide cassette comprising         the first, second, and third nucleotide sequences oriented 5′ to         3′ as:         -   (i) the first nucleotide sequence, wherein the first             nucleotide sequence is a forward primer binding site,         -   (ii) the second nucleotide sequence, wherein the second             nucleotide sequence is a barcode, and         -   (iii) the third nucleotide sequence, wherein the third             nucleotide sequence is a reverse primer binding site;     -   (d) inserting the native barcode polynucleotide cassette into         the genome of a host cell of the same genus as the donor cell in         (a); and     -   (e) repeating (d) one or more times with a different host cell         of the same genus each time; wherein the nucleotide sequences of         (i), (ii), and (iii) are native to the host cell, thereby         barcoding a population of host cells.         131. The method of embodiment 130, wherein the population of         host cells share a common species.         132. The method of embodiment 130, wherein each of the different         host cells of (e) is of a different strain.         133. The method of embodiment 130, wherein each of the different         host cells of (e) is of a different species.         134. The method of any one of embodiments 130-133, wherein the         barcode comprises a constant barcode region and a variable         barcode region.         135. The method of embodiment 134, wherein the constant barcode         region of the barcode inserted into each host cell of the         population of host cells is the same in each of the host cells.         136. The method of embodiment 134 or 135, wherein the variable         barcode region of the barcode inserted into each host cell of         the population of host cells is different, with only host cells         of the same strain, species, or other categorical distinction         sharing the same variable region.         137. The method of any one of embodiments 130-136, wherein the         insertion of the cassette into the genome of the host cell         introduces one or more stop codons in either orientation in the         host cell.         138. The method of any one of embodiments 130-137, wherein the         cassette is present in the genome of the host cell between two         coding regions separated by a terminator region.         139. The method of embodiment 138, wherein the cassette is         present between the terminator region and one of the two coding         regions.         140. The method of any one of embodiments 130-139, wherein the         cassette is present in the genome of the host cell at a set         distance from an origin of replication.         141. The method of embodiment 140, wherein the set distance from         the origin of replication is similar or identical in each host         cell of the population of host cells.         142. A composition comprising:     -   (a) a population of modified cells comprising, in their genome.         -   (i) a naturally occurring first nucleotide sequence, second             nucleotide sequence, and third nucleotide sequence, and         -   (ii) a native barcode polynucleotide cassette comprising the             first, second, and third nucleotide sequences oriented 3′ to             3′ as:             -   (1) the first nucleotide sequence, wherein the first                 nucleotide sequence is a forward primer binding site,             -   (2) the second nucleotide sequence, wherein the second                 nucleotide sequence is a barcode, and             -   (3) the third nucleotide sequence, wherein the third                 nucleotide sequence is a reverse primer binding site;                 wherein the first, second, and third nucleotide                 sequences of (ii) are in a different proximity to one                 another as compared to the naturally occurring sequences                 of (i).                 143. The composition of embodiment 142, wherein the                 population of modified cells share a common species.                 144. The composition of embodiment 142, wherein the                 population of modified cells comprises more than one                 species.                 145. The composition of embodiment 144, wherein the                 population of modified cells comprises more than one                 strain of each species.                 146. The composition of any one of embodiments 142-145,                 wherein the barcode comprises a constant barcode region                 and a variable barcode region.                 147. The composition of embodiment 146, wherein the                 constant barcode region is the same in each of the                 modified cells.                 148. The composition of embodiment 146 or 147, wherein                 the variable barcode region in the population of                 modified cells is not conserved, with only modified                 cells of the same strain, species, or other categorical                 distinction sharing the same variable region.                 149. The composition of any one of embodiments 142-148,                 wherein as a result of the presence of the cassette, the                 population of modified cells comprise one or more stop                 codons in either orientation in the modified cells.                 150. The composition of any one of embodiments 142-149,                 wherein the cassette is present in the genome of the                 host cell between two coding regions separated by a                 terminator region.                 151. The composition of embodiment 150, wherein the                 cassette is present between the terminator region and                 one of the two coding regions.                 152. The composition of any one of embodiments 142-151,                 wherein the cassette is present in the genome of the                 host cell at a set distance from an origin of                 replication.                 153. The composition of embodiment 152, wherein the set                 distance from the origin of replication is similar or                 identical in each modified cell of the population of                 modified cells.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world. Further, U.S. Pat. No. 9,975,817, issued on May 22, 2018, and entitled: Methods and Compositions for Improving Plant Traits, is hereby incorporated by reference. Further, PCT/US2018/013671, filed Jan. 12, 2018, published as WO2018132774A1 on Jul. 19, 2018, and entitled: Methods and Compositions for Improving Plant Traits, are hereby incorporated by reference. Further, PCT/US2019/041429, filed Jul. 11, 2019, and entitled: Temporally and Spatially Targeted Dynamic Nitrogen Delivery by Remodeled Microbes, is hereby incorporated by reference. 

1.-38. (canceled)
 39. A composition comprising: (a) one or more modified cells comprising, in their genome: (i) a naturally occurring first nucleotide sequence, second nucleotide sequence, and third nucleotide sequence, and (ii) a native barcode polynucleotide cassette comprising the first, second, and third nucleotide sequences oriented 5′ to 3′ as: (1) the first nucleotide sequence, wherein the first nucleotide sequence is a forward primer binding site, (2) the second nucleotide sequence, wherein the second nucleotide sequence is a barcode, and (3) the third nucleotide sequence, wherein the third nucleotide sequence is a reverse primer binding site; wherein the first, second, and third nucleotide sequences of (ii) are in a different proximity to one another as compared to the naturally occurring sequences of (i).
 40. The composition of claim 39, wherein the barcode is unique to different species of the one or more modified cells.
 41. The composition of claim 39, wherein the barcode is unique to different strains of the one or more modified cells. 42.-47. (canceled)
 48. The composition of claim 39, wherein the one or more modified cells is a bacterial cell.
 49. The composition of claim 39, wherein the nucleotide sequence of at least one of (1), (2), or (3) is isolated from ribosomal DNA or internal transcribed spacer (ITS) DNA.
 50. The composition of claim 39, wherein the nucleotide sequence of at least one of (1), (2), or (3) is 16S rDNA or 18S rDNA.
 51. The composition of claim 39, wherein at least one of the primer binding sites is selected from 8F, 27F, CCF, 357F, 515F, 533F, 16S.1100.F16, 804F, 1237F, 338R, 519R, CDR, 806R, 907R, 1100R, 1391R, 1392R, 1492R(l), and 1492R(s).
 52. The composition of claim 39, wherein the naturally occurring first and third nucleotide sequences in the genome of the modified cells occur in the same orientation as one another.
 53. The composition of claim 39, wherein at least one of the naturally occurring first, second, or third nucleotide sequences do not occur immediately adjacent to one another in the genome of the modified cell. 54.-56. (canceled)
 57. The composition of claim 39, wherein the barcode comprises a constant barcode region and a variable barcode region.
 58. The composition of claim 57, wherein the constant barcode region of the barcode is the same in each of the one or more modified cells.
 59. The composition of claim 57, wherein the variable barcode region of the barcode is different in each of the one or more modified cells, with only cells of the same strain, species, or other categorical distinction sharing the same variable region. 60.-64. (canceled)
 65. The composition of claim 48, wherein the one or more modified bacteria are transgenic.
 66. The composition of claim 48, wherein the one or more modified bacteria are non-intergeneric remodeled bacteria. 67.-68. (canceled)
 69. The composition of claim 66, wherein the non-intergeneric remodeled bacteria comprise, or are derived from, a bacterium selected from Table
 1. 70. (canceled)
 71. The composition of claim 48, wherein the bacterial cell is capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.
 72. The composition of claim 48, wherein the bacterial cell comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network.
 73. The composition of claim 48, wherein the bacterial cell comprises an introduced control sequence operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
 74. The composition of claim 48, wherein the bacterial cell comprises a heterologous promoter operably linked to at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
 75. The composition of claim 48, wherein the bacterial cell comprises at least one genetic variation introduced into a member selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, a gene associated with biosynthesis of a nitrogenase enzyme, and combinations thereof.
 76. The composition of claim 48, wherein the bacterial cell comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; and decreased uridylyl-removing activity of GlnD. 77.-79. (canceled)
 80. The composition of claim 48, wherein the bacterial cell comprises at least one of: a mutated nifL gene comprising a heterologous promoter in said nifL gene; a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; and combinations thereof. 81.-82. (canceled)
 83. The composition of claim 48, wherein the bacterial cell is selected from Rahnella aquatilis, Klebsiella variicola, Achromobacter spiritinus, Achromobacter marplatensis, Microbacterium murale, Kluyvera intermedia, Kosakonia pseudosacchari, Enterobacter sp., Azospirillum lipoferum, and Kosakonia sacchari.
 84. The composition of claim 48, wherein the bacterial cell is endophytic, epiphytic, or rhizospheric.
 85. The composition of claim 48, wherein the bacterial cell is selected from: a bacterium deposited as NCMA 201701002, a bacterium deposited as NCMA 201708004, a bacterium deposited as NCMA 201708003, a bacterium deposited as NCMA 201708002, a bacterium deposited as NCMA 201712001, and a bacterium deposited as NCMA
 201712002. 86.-153. (canceled) 